Detecting esophageal diseases

Abstract

The present invention relates to detecting esophageal diseases. Provided herein are techniques for screening for esophageal diseases, particularly but not exclusively, methods, compositions, and related uses for detecting the presence of esophageal diseases (e.g., Barrett's esophagus, Barrett's esophagus dysplasia, etc.). In addition, the present technology provides methods, compositions, and related uses for distinguishing between Barrett's esophagus and Barrett's esophagus dysplasia, as well as low-grade dysplasia, high-grade dysplasia, and esophageal adenocarcinoma in samples obtained by endoscopic brushing or non-endoscopic whole esophageal brushing or swabbing using a tethered device (e.g., a capsule sponge, balloon, or other device).

Description

[0001] This application is a divisional application of Chinese patent application 201680018938.3 “Detection of esophageal disease” filed on March 23, 2016. Technical Field

[0002] Provided herein are techniques for screening for esophageal diseases, particularly, but not exclusively, methods, compositions, and related uses for detecting the presence of esophageal diseases, such as Barrett's esophagus, Barrett's esophageal dysplasia, and the like. Furthermore, the present technology provides methods, compositions, and related uses for distinguishing between Barrett's esophagus and Barrett's esophageal dysplasia, as well as low-grade dysplasia, high-grade dysplasia, and esophageal adenocarcinoma in samples obtained by endoscopic brushing or non-endoscopic whole esophageal brushing or swabbing using a tethered device, such as a capsule sponge, balloon, or other device. Background Art

[0003] In Barrett's esophagus, healthy esophageal epithelium is replaced by metaplastic columnar cells, thought to be the result of damage to the esophagus from long-term exposure to reflux from gastroesophageal reflux disease (GERD). An inherent risk of progression from Barrett's esophagus to esophageal adenocarcinoma has been established. Histologically, this progression includes a clear sequential stage from isolated metaplasia to low-grade dysplasia, then to high-grade dysplasia, and finally to adenocarcinoma.

[0004] The 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 can prevent subsequent transformation to cancer. This dysplasia is generally indistinguishable from nondysplastic Barrett's esophagus using endoscopy, and periodic random biopsies and histologic evaluation are the current surveillance approach for patients with proven Barrett's esophagus.

[0005] There is little evidence to support the hypothesis that antisecretory agents or antireflux surgery prevent the development of adenocarcinoma or cause regression of Barrett's esophagus (see, eg, 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 the treatment of GERD. However, some studies using cimetidine or ranitidine did not document resolution 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 regression of Barrett's esophagus was not optimistic, and studies conducted in this area have been inconclusive to date. Only two of seven investigators demonstrated some regression. Most were unable to detect any regression, despite complete normalization of esophageal pH as demonstrated by pH testing.

[0008] Currently, the indications for medical treatment of Barrett's esophagus—symptom control and healing of the esophageal mucosa—are the same as those for GERD.

[0009] Barrett's esophagus is the precursor lesion for most esophageal adenocarcinomas, malignancies with rapidly increasing incidence and persistently poor outcomes. As previously mentioned, early detection of esophageal adenocarcinoma has been shown to be associated with earlier stages and increased survival. Furthermore, detection of dysplasia with subsequent endoscopic ablation may prevent esophageal adenocarcinoma.

[0010] There is a clear need for improved methods for detecting Barrett's esophagus and related diseases, such as Barrett's esophagus dysplasia. Summary of the Invention

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

[0012] When applied to animal models and human cell lines, the analysis of CpG islands 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:e1000438). Furthermore, methylation levels were bimodally distributed between highly methylated and unmethylated sequences, further supporting a binary switch-like pattern of DNA methyltransferase activity (Zhang et al. (2009) PLoS Genet 5:e1000438). Analysis of in vitro mouse tissues and cell lines showed that only approximately 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. Among them, HCP sites with >50% methylation are multiple established markers, such as Wnt 2, NDRG2, SFRP2, and BMP3 (Meissner et al. (2008) Nature 454:766–70).

[0013] Thus, provided herein are techniques for screening (e.g., monitoring) of esophageal diseases, particularly, but not exclusively, methods, compositions, and related uses for detecting the presence of esophageal diseases (e.g., Barrett's esophagus, Barrett's esophagus dysplasia, etc.). In addition, the present technology provides methods, compositions, and related uses for distinguishing between Barrett's esophagus and Barrett's esophagus dysplasia, as well as low-grade dysplasia of Barrett's esophagus, high-grade dysplasia of Barrett's esophagus, and esophageal adenocarcinoma in samples obtained by endoscopic brushing or non-endoscopic whole esophageal brushing or swabbing using a tethered device (e.g., a capsule sponge, balloon, or other device).

[0014] In fact, experiments conducted during the development of the present technology compared the methylation status of DNA markers from esophageal tissue of subjects with Barrett's esophagus with the methylation status of the same DNA markers from control subjects (e.g., normal tissue of each tissue type) and with the methylation status of the same DNA markers from subjects with Barrett's esophagus dysplasia (see Examples 1 and 5).

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

[0016] Markers and / or marker panels (eg, chromosomal regions with annotations provided in Tables 2, 3, 5, and / or 6) capable of classifying BE and Barrett's esophagus-related dysplasia (BED) in esophageal tissue were identified (see Examples 1, 3, and 4).

[0017] Markers and / or marker panels (e.g., chromosomal regions with annotations provided in Table 5) were identified that are capable of predicting Barrett's esophagus-associated low-grade dysplasia (BE-LGD), Barrett's esophagus-associated dysplasia-high-grade dysplasia (BE-HGD), and esophageal adenocarcinoma (EAC) in samples obtained by whole esophageal swab or brushing (see Examples 1 and 4).

[0018] Markers and / or marker panels (eg, chromosomal regions with annotations provided in Table 5) were identified that are capable of classifying BE and BED in samples obtained by whole esophageal swab or brushing (see Examples 1 and 4).

[0019] As described herein, the present technology provides a number of methylated 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) with high discriminatory power for esophageal diseases (e.g., BE, BED, BE-LGD, BE-HGD, EAC). The experiments applied selection filters for candidate markers to identify markers that provide a high signal-to-noise ratio and low background levels, such as to provide high specificity when assaying a medium (e.g., esophageal tissue) for screening or diagnostic (e.g., cancer screening or diagnosis) purposes.

[0020] In some embodiments, the present technology relates to the presence and methylation state of one or more markers identified herein in the assessment biological sample. These markers include one or more differential methylation regions (DMRs) as discussed herein, for example, as provided in Tables 1, 2, 3, 5, 6, 7, and 8. In the embodiments of the present technology, methylation state is assessed. Therefore, the technology provided by the present invention does not limit the method for measuring the methylation state of a gene. For example, in some embodiments, methylation state is measured by genome scanning methods. For example, one method relates to restriction marker genome scanning (Kawai et al. (1994) Mol. Cell. Biol. 14: 7421–7427), and another example relates to methylation sensitivity arbitrary primer PCR (Gonzalgo et al. (1997) Cancer Res. 57: 594–599). In some embodiments, by digesting genomic DNA with methylation sensitivity restriction enzymes, the target region is then subjected to Southern analysis (digestion-Southern method) to monitor the change in the methylation pattern of a specific CpG site. In some embodiments, analyzing 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 utilizing bisulfite-treated DNA as a starting point for methylation analysis have been reported. 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 detecting gene mutations (Kuppuswamy et al. (1991) Proc. Natl. Acad. Sci. USA 88: 1143–1147) and for quantification 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 PCR-generated template and terminate immediately 5' of the single nucleotide to be assayed. In some embodiments, the method using the "quantitative Ms-SNuPE assay" described in U.S. Patent No. 7,037,650 is used.

[0021] When assessing methylation status, methylation status is typically expressed as the fraction or percentage of a single strand of DNA methylated at a specific site (e.g., at a single nucleotide, at a specific region or locus, at a longer target sequence, e.g., up to a DNA subsequence of about 100 bp, 200 bp, 500 bp, 1000 bp or longer) relative to the total population of DNA in a sample comprising the specific site. Typically, the amount of unmethylated nucleic acid is determined by PCR using a calibrator. Then, a known amount of DNA is treated with bisulfite, and the resulting methylation-specific sequence is determined using real-time PCR or other exponential amplification (e.g., QuARTS assay).

[0022] For example, in some embodiments, the method includes generating a standard curve for an unmethylated target by using an external standard. The standard curve is constructed by at least two points, and the real-time Ct value of the unmethylated DNA is associated with a known quantitative standard. Then, a second standard curve for the methylated target is constructed by at least two points and an external standard. This second standard curve associates the Ct of the methylated DNA with a known quantitative standard. Next, the Ct values ​​of the methylated and unmethylated populations of the test sample are determined, and the genomic equivalent of the DNA is calculated by the standard curves generated in the first two steps. The percentage of methylation at the target site is calculated by the amount of methylated DNA relative to the total amount of DNA in the population, for example (number of methylated DNA) / (number of methylated DNA+number of unmethylated DNA)×100.

[0023] Also provided herein are compositions and kits for implementing the method. For example, in some embodiments, there is provided a reagent (for example, primer, probe) specific to one or more markers, which is separate or in groups (for example, a primer pair group for amplifying a variety of markers). Other reagents for detecting assays (for example, enzymes, buffers, positive and negative controls for carrying out QuARTS, PCR, sequencing, bisulfite or other assays) can also be provided. In some embodiments, there is provided a kit comprising one or more reagents necessary, sufficient or useful for carrying out the method. Also provided is a reaction mixture comprising reagents. Also provided is a main mixed reagent group comprising a variety of reagents that can be added to each other and / or added to a test sample to complete a reaction mixture.

[0024] In some embodiments, the technology described herein is associated with a programmable machine designed to perform a series of arithmetic or logical operations provided by the methods described herein. For example, some embodiments of the present technology are associated with (e.g., executed in) computer software and / or computer hardware. On the one hand, the technology relates to a computer that includes a form of memory, an element for performing arithmetic and logical operations, and a processing element (e.g., a microprocessor) for executing a series of instructions (e.g., the 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 provided in Table 8). 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); 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 provided in Table 5 77, 90, and 135; 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 Ct values; calculating the score, frequency, or percentage of methylation (e.g., methylation of one or more DMRs, such as DMRs 1-78 provided in Table 1; DMRs provided in Table 2 3, 5, 30, 33, 43, 58, 77, and 79-128; 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); identifying CpG islands; determining the specificity and / or sensitivity of an assay or marker; calculating ROC curves and associated AUCs; and sequence analysis; all as described herein or known in the art.

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

[0026] In some embodiments, a 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 the multiple assays (e.g., determining the methylation status of multiple DMRs, such as the DMRs provided in Tables 1, 2, 3, 5, 6, 7, 8) to report to a user. Related embodiments calculate risk factors based on a mathematical combination (e.g., a weighted combination, a linear combination) of the results from multiple assays (e.g., determining the methylation status of multiple markers (e.g., determining the methylation status of multiple DMRs, such as the DMRs provided in Tables 1, 2, 3, 5, 6, 7, 8)). In some embodiments, the methylation status of the DMRs defines a dimension and can have values ​​in a multidimensional space and the coordinates defined by the methylation status of the multiple DMRs are, for example, results related to esophageal disease risk (e.g., risk of BE, BED, BE-LGD, BE-HGD, EAC) reported to the user.

[0027] Some embodiments include storage media and memory components. The memory components (e.g., volatile and / or non-volatile memory) are used to store instructions (e.g., embodiments of the processes provided herein) and / or data (e.g., artifacts, such as methylation measurements, sequences, and statistical descriptions associated therewith). 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 existing technologies as well as technologies under development or yet to be developed (e.g., quantum computers, chemical computers, DNA computers, optical computers, spintronic-based computers, etc.).

[0029] In some embodiments, the present technology includes wired (e.g., metal cables, optical fibers) or wireless transmission media for transmitting data. For example, some embodiments involve the transmission of data 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, and in some embodiments, the programmable machines have a client / server relationship.

[0030] In some embodiments, the data is stored on a computer-readable storage medium, such as a hard disk, flash memory, optical media, floppy disk, or the like.

[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, such as in the implementation of cluster computing or grid computing or some other distributed computer architecture that relies on a complete computer (with onboard CPU, memory, power supply, network interface, etc.) connected to a network (private, public or Internet) via conventional network interfaces such as Ethernet, fiber optic or wireless networking technology.

[0032] For example, some embodiments provide a computer including a computer-readable medium. The embodiment includes a random access memory (RAM) coupled to a processor. The processor executes computer-executable program instructions stored in the memory. Such a processor may include a microprocessor, an ASIC, a state machine, or other processor, and may be any one of a plurality of computer processors, such as a processor from Intel Corporation of Santa Clara, California, and a processor from Motorola Corporation of Schaumburg, Illinois. Such a processor includes or can communicate with a medium, such as a computer-readable medium, which 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 that can provide computer-readable instructions to the processor. Other examples of suitable media include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROMs, RAMs, ASICs, configured processors, all optical media, all tapes or other magnetic media, or any other medium from which a computer processor can read instructions. In addition, various other forms of computer-readable media can send or carry instructions to a computer, including wired and wireless routers, private or public networks or other transmission devices or channels. 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, the computer is connected to a network. The computer can also include many external or internal devices, such as a mouse, CD-ROM, DVD, keyboard, display or other input or output devices. The example of a computer is a personal computer, digital assistant, personal digital assistant, cellular phone, mobile phone, smart phone, pager, digital tablet computer, laptop computer, internet equipment and other devices based on processors. Generally, the computer relevant to the aspect of the technology provided herein can be any type of platform based on processors, which can support any operating system (such as, Microsoft Windows, Linux, UNIX, Mac OS X etc.) that comprises one or more programs of the technology provided herein. Some embodiments include the personal computer that performs other application programs (such as, application software). Application programs can be included in a memory, and can include, for example, word processing applications, electronic spreadsheet applications, email applications, instant messaging applications, presentation applications, internet browser applications, calendar / manager applications and any other application that can be performed by a client device.

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

[0036] Thus, provided herein are techniques related to methods of screening for BE in a sample obtained from a subject, the methods comprising determining the methylation state of a marker in a sample obtained from the subject; and identifying the subject as having BE when the methylation state of the marker is different from the methylation state of the marker determined in a subject that does not have BE, wherein the marker comprises one or more bases 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 provided in Table 8 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.

[0037] Provided herein are technologies relating to methods of distinguishing BE from BED in a sample obtained from a subject, the methods comprising determining the methylation state of a marker in a sample obtained from a subject; and identifying the subject as having BE when the methylation state of the marker is similar to the methylation state of the marker determined in a subject with BE, or identifying the subject as having BED when the methylation state of the marker is similar to the methylation state of the marker determined in a subject with 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 relating to methods of distinguishing BE-LGD, BE-HGD, and BE-EAC in a sample obtained from a subject, the methods comprising determining the methylation state of a marker in a sample obtained from a subject; and identifying the subject as having BE-LGD when the methylation state of the marker is similar to the methylation state of the marker determined in a subject with BE-LGD, identifying the subject as having BE-HGD when the methylation state of the marker is similar to the methylation state of the marker determined in a subject with BE-HGD, or identifying the subject as having EAC when the methylation state of the marker is similar to the methylation state of the marker determined in a subject with 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 state assessed. In some embodiments, assessing the methylation state of a marker in a sample comprises determining the methylation state of a base. In some embodiments, determining the methylation state of a marker in a sample comprises determining the degree of methylation of multiple bases. Furthermore, in some embodiments, the methylation state of a marker comprises elevated methylation of the marker relative to a normal methylation state of the marker. In some embodiments, the methylation state of a marker comprises decreased methylation of the marker relative to a normal methylation state of the marker. In some embodiments, the methylation state of a marker comprises a different methylation pattern of the marker relative to a normal methylation state of the marker.

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

[0041] This technology is not limited by sample type. In some embodiments, the sample is the esophageal tissue (see Example 1 and Table 5) (see Example 5 and Table 8) obtained by wiping or brushing the whole esophagus. In some embodiments, the sample is the esophageal tissue (see Example 5 and Table 8) obtained by using a sponge capsule device. For example, in some embodiments, the sample is a fecal sample, a tissue sample (such as esophageal tissue, gastric tissue, pancreatic tissue, bile duct / liver tissue and colorectal tissue), a blood sample (such as blood plasma, serum, whole blood), excreta or urine sample.

[0042] In addition, the present technology is not limited to the method for determining methylation status. In some embodiments, determination includes the use of methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, separation based on mass or target capture. In some embodiments, determination includes the use of methylation-specific oligonucleotides. In some embodiments, the present technology uses large-scale parallel sequencing (e.g., next generation sequencing) to determine methylation status, such as by synthetic sequencing, real-time (e.g., single molecule) sequencing, bead emulsion sequencing, nanopore sequencing, etc.

[0043] The present technology provides reagents for detecting DMRs, for example, in some embodiments, sets of oligonucleotides comprising sequences provided as SEQ ID NOs: 1 to 50 are provided. In some embodiments, oligonucleotides comprising sequences complementary to chromosomal regions having bases in DMRs are provided, for example, oligonucleotides sensitive to the methylation status of DMRs.

[0044] The present technology provides a plurality of marker panels, for example, in some embodiments, the markers comprise a chromosomal region having an annotation provided in Tables 1, 2, 3, 5, 6, 7 and / or 8, and include markers (see, Tables 1, 2, 3, 5, 6, 7, 8). In addition, embodiments provide methods for analyzing one or more DMRs from DMR No. 1-229 of Tables 1, 2, 3, 5, 6, 7 and / or 8.

[0045] Kit embodiments are provided, e.g., a kit comprising: 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 state associated with a subject without esophageal disease (e.g., a subject without BE, BED, BE-LGD, BE-HGD, and EAC). In some embodiments, the kit comprises a bisulfite reagent and an oligonucleotide as 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 state associated with a subject with esophageal disease (e.g., a subject with BE) (e.g., a subject with BED, BE-LGD, BE-HGD, and EAC). Some kit embodiments comprise a sample collector for obtaining a sample (eg, a stool sample) from a subject; reagents for isolating nucleic acids from the sample; a bisulfite reagent; and an oligonucleotide described herein.

[0046] The present technology is related to embodiments of compositions (e.g., reaction mixtures). In some embodiments, compositions are provided, comprising a nucleic acid containing a DMR and a bisulfite reagent. Some embodiments provide compositions, comprising a nucleic acid containing a DMR and an oligonucleotide as described herein. Some embodiments provide compositions, comprising a nucleic acid containing a DMR and a methylation-sensitive restriction enzyme. Some embodiments provide compositions, comprising a nucleic acid containing a DMR and a polymerase.

[0047] Provided are embodiments of additional related methods for screening for esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC) in a sample obtained from a subject, for example, the method comprising determining the methylation state 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 state of the marker in the sample from the subject with the methylation state of the marker in a normal control sample from a subject without esophageal disease; and determining a confidence interval and / or p-value for the difference in methylation state between the sample from the subject 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 following steps: 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 the nucleotide sequence of a nucleic acid comprising a DMR from a subject who does not have cancer to identify a difference in the two sequences; and identifying the subject as having a tumor when a difference is present.

[0048] The present technology provides a system 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, for example, a system for screening for esophageal disease in a sample obtained from a subject, the system comprising: an analysis component configured to determine the methylation state of the sample; a software component configured to compare the methylation state of the sample with the methylation state of a control sample or reference sample recorded in a database; and an alarm component configured to alert a user of a methylation state associated with an esophageal disease (e.g., a methylation state in the absence of esophageal disease; a methylation state of BE; a methylation state of BED; a methylation state of BE-LGD; a methylation state of BE-HGD; a methylation state of EAC). In some embodiments, the alarm is determined by a software component that receives results from multiple assays (e.g., determining the methylation state of multiple markers, such as the DMRs provided in 1, 2, 3, 5, 6, 7, and / or 8) and calculates a value or result to be reported based on the multiple results. Some embodiments provide a database of weighted parameters associated with each DMR provided herein for use in calculating a value or outcome and / or alert for reporting to a user (e.g., physician, nurse, clinician, etc.). In some embodiments, all results from multiple assays are reported, and in some embodiments, one or more results are used to provide a score, value, or outcome that indicates the risk of esophageal disease in a subject (e.g., indicating the risk of BE; indicating the risk of BED; indicating the risk of BE-LGD; indicating the risk of BE-HGD; indicating the risk of EAC) based on the integration of one or more results from multiple assays.

[0049] In some embodiments of the system, the sample comprises a nucleic acid containing a DMR. In some embodiments, the system further comprises a component for isolating nucleic acids, a component for collecting samples, such as a component for collecting fecal samples. In some embodiments, the system comprises a nucleic acid sequence containing a DMR. In some embodiments, the database comprises nucleic acid sequences from subjects without esophageal disease. Nucleic acids are also provided, such as a group of nucleic acids, each nucleic acid having a sequence comprising a DMR. In some embodiments, a group of nucleic acids, each nucleic acid having a sequence from a subject without esophageal disease. Related system embodiments include a group of nucleic acids as described and a database of nucleic acid sequences associated with the group of nucleic acids. Some embodiments further comprise a bisulfite reagent. Moreover, some embodiments further comprise a nucleic acid sequencer.

[0050] In certain embodiments, a method for detecting Barrett's esophagus in a sample obtained from a subject is provided, 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 treated in step b), wherein the one or more DNA methylation markers comprise a DNA methylation marker selected from the group consisting of 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 with a reference methylation level of the one or more DNA methylation markers of the following subjects: i) a subject who does not have Barrett's esophagus, to identify a difference in the two sequences, and ii) a subject who does not have Barrett's esophagus dysplasia, to identify a difference in the two sequences; and e) when the difference in i) and ii) is present, identifying the subject as having Barrett's esophagus.

[0051] In certain embodiments, a method for detecting Barrett's esophagus dysplasia in a sample obtained from a subject is provided, 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; and c) determining the methylation level of one or more DNA methylation markers in the DNA treated in step b), wherein the one or more DNA methylation markers comprise a methylation marker selected from a DMR. No. 3, 5, 30, 33, 43, 58, 77, 79-187, d) comparing the determined methylation levels of the one or more DNA methylation markers with the methylation level references of the one or more DNA methylation markers of the following subjects: i) a subject with Barrett's esophagus to identify the difference in the two sequences, and ii) a subject without Barrett's esophagus dysplasia to identify the difference in the two sequences; and e) when the difference in i) and ii) exists, identifying the subject as having Barrett's esophagus dysplasia.

[0052] In certain embodiments, a method for detecting low-grade dysplasia of Barrett's esophagus in a sample obtained from a subject is provided, 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 differentially methylated regions (DMRs) provided by DMR Nos. 77, 90, and 135; and d) converting the one or more DNA methylation markers into bases in a differentially methylated region (DMR) provided by DMR Nos. 77, 90, and 135. The determined methylation level of the one or more DNA methylation markers is compared to a reference methylation level of the one or more DNA methylation markers of: i) a subject without low-grade dysplasia of Barrett's esophagus, to identify a difference in the two sequences, ii) a subject without dysplasia of Barrett's esophagus, to identify a difference in the two sequences, iii) a subject with high-grade dysplasia of Barrett's esophagus, to identify a difference in the two sequences, and iv) a subject with esophageal adenocarcinoma, to identify a difference in the two sequences; and e) identifying the subject as having low-grade dysplasia of Barrett's esophagus when a difference in i), ii), iii) and iv) is present.

[0053] In certain embodiments, a method for detecting high-grade dysplasia of Barrett's esophagus in a sample obtained from a subject is provided, 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 differentially methylated regions (DMRs) provided by DMR Nos. 77, 90, and 135; d) converting the one or more DNA methylation markers into bases in a differentially methylated region (DMR) provided by DMR Nos. 77, 90, and 135; The determined methylation levels of the plurality of DNA methylation markers are compared to a reference of methylation levels of the one or more DNA methylation markers of: i) a subject without high-grade dysplasia of Barrett's esophagus to identify a difference in the two sequences, ii) a subject without dysplasia of Barrett's esophagus to identify a difference in the two sequences, iii) a subject with low-grade dysplasia of Barrett's esophagus to identify a difference in the two sequences, and iv) a subject with esophageal adenocarcinoma to identify a difference in the two sequences; and e) identifying the subject as having high-grade dysplasia of Barrett's esophagus when a difference in i), ii), iii, and iv) is present.

[0054] In certain embodiments, a method for detecting esophageal adenocarcinoma in a sample obtained from a subject is provided, comprising a) obtaining a sample comprising DNA from the subject; 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; and c) determining the methylation level of one or more DNA methylation markers in the DNA treated in step b), wherein the one or more DNA methylation markers comprise a methylation marker selected from a group consisting of a DMR. Nos. 77, 90 and 135, d) comparing the determined methylation levels of the one or more DNA methylation markers with reference methylation levels of the one or more DNA methylation markers of: i) a subject without esophageal adenocarcinoma to identify the difference between the two sequences, ii) a subject without Barrett's esophagus dysplasia to identify the difference between the two sequences, iii) a subject with low-grade dysplasia of Barrett's esophagus to identify the difference between the two sequences, and iv) a subject with high-grade dysplasia of Barrett's esophagus to identify the difference between the two sequences; and e) identifying the subject as having esophageal adenocarcinoma when the difference in i), ii), iii and iv) exists.

[0055] In some embodiments, determination of elevated methylation in one or more DNA methylation markers comprises determination of altered methylation in regions selected from CpG islands and CpG island shores.

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

[0057] In some embodiments, determining the methylation level of one or more DNA methylation markers in the DNA 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 in step b) is performed by bisulfite modification of the obtained DNA.

[0058] In some embodiments, determining 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 whole esophageal brushing or swabbing using a tethered device (e.g., a capsule sponge, balloon, or other device).

[0060] Additional embodiments will be apparent to persons skilled in the relevant art(s) based on the teachings contained herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 : Positive rates of a panel of three markers (DIO3, MAX20.218, NDRG4) (Table 5) in tissue DNA from BE subgroups without dysplasia and with dysplasia of varying severity (Examples I, III, III, and IV).

[0062] Figure 2 : Methylated DNA marker levels (PCR copies / 20 ng DNA) in BE cases and normal (N1) controls from Phase 2 (endoscopic brush study) (Example V).

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

[0064] Figure 4 : Methylated DNA marker levels (PCR copies / 30 ng DNA) in BE cases and normal (N1) controls from Phase 3 (capsule sponge study) (Example V).

[0065] Details

[0066] Barrett's esophagus (BE) is the strongest risk factor and the only known precursor to esophageal adenocarcinoma (EAC), a lethal malignancy with a poor survival rate (<20% at 5 years) when detected after symptom onset (see Nelsen EM, et al., The Surgical clinics of North America 2012; 92: 1135-54). The incidence of esophageal adenocarcinoma has increased by nearly 600% in the population over the past three decades (see, Hur C, et al., Cancer 2013; 119: 1149-58). BE progresses in a stepwise manner from no dysplasia, to low-grade dysplasia (LGD), to high-grade dysplasia (HGD) to cancer to EAC. This sequence of metaplasia to dysplasia to carcinoma has prompted gastroenterological societies in several countries to recommend screening for BE in high-risk patients with multiple risk factors, followed by endoscopic surveillance (depending on the grade of dysplasia) to detect the progression of dysplasia or carcinoma at an early stage (see Spechler SJ, et al., Gastroenterology 2011;140:e18-52; Wang KK, et al., AmJ Gastroenterol 2008;103:788-97; Fitzgerald RC, et al., Gut 2014;63:7-42). Endoscopic treatment of LGD, HGD, and early-stage cancer has been developed and shown to be effective in reducing the incidence of cancer and improving survival in subjects with BE (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 that the normal squamous lining of the esophagus is replaced by metaplastic columnar epithelium in patients with BE. However, sedated endoscopy is expensive in terms of direct and indirect costs and is not suitable for widespread use. 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 and lower cost to sEGD, but are still less widely accepted by providers as a 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, its use by consulting physicians remains limited (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 widely applicable 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 carefully examining the BE segment using high-resolution white light imaging and advanced imaging techniques, endoscopic screening for dysplasia is currently performed using four-quadrant random biopsies of the BE segment every 1-2 cm. Although 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 actual gastroenterologists (see, Abrams JA, et al., Clin Gastroenterol Hepatol 2009). In fact, compliance decreases with increasing BE segment length, leading to an increased incidence of false dysplasia. Other challenges in detecting dysplasia 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 interobserver agreement between pathologists in grading dysplasia, and the relatively poor sensitivity of current surveillance strategies in detecting generalized dysplasia or cancer (see Sharma P, et al., Gastroenterology 2004; 127: 310-30). The use of advanced imaging technologies in the community remains unclear, with only one-third of practicing gastroenterologists reporting regular use in BE monitoring (see Singh M, et al., Gastrointestinal endoscopy 2013; 78: 689-95). A sponge on a string device has recently been studied 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 connected to a string. The capsule is swallowed by the patient. The gelatin shell of the capsule dissolves in gastric fluid, releasing the foam device as a sphere, which is then pulled out with an attached string, providing a brushing / cell sample of the proximal stomach and esophagus. These samples can then be studied for biomarkers to detect BE. Two large multicenter studies have been conducted in the UK using this device, using trefoil factor 3 (a protein specific for BE epithelial cells) detected in immunohistochemistry as a BE marker, 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). The sensitivity and specificity of this marker in detecting BE were reported to be 73% and 94% for BE segments with a circumference of >1 cm.In addition, this capsule sponge device has been safely used in a study conducted 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). Methylated 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: 272-7 e1).

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

[0070] With respect to the technology described herein, the section headings used are for organizational purposes only and are not to be construed as limiting the subject matter in any way.

[0071] In the detailed description of each embodiment, for the purpose of illustration, many specific details are set forth to provide a thorough understanding of the disclosed embodiments. However, it will be understood by those skilled in the art that these various embodiments can be implemented with or without these specific details. In other cases, structures and devices are shown in block diagram form. In addition, it will be readily understood by those skilled in the art that the specific order in which the methods are presented and implemented is illustrative, and it is contemplated that the order may vary and still remain within the spirit and scope of the various embodiments disclosed herein.

[0072] All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers and internet web pages, are incorporated herein by reference in their entirety for the purposes described. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the various embodiments described herein belong. When the definition of a term in an incorporated reference appears to differ from the definition provided in this teaching, the definition provided in this teaching shall prevail.

[0073] definition

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

[0075] Throughout the specification and claims, the following terms have the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used herein does not necessarily refer to the same embodiment, although it may be. Additionally, the phrase "in another embodiment" as used herein does not necessarily refer to a different embodiment, although it may be. Thus, as described below, the various embodiments of the present invention may be readily combined without departing from the scope or spirit of the invention.

[0076] In addition, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described unless the context clearly dictates otherwise. In addition, throughout this specification, the meanings of "a," "an," and "the" include plural referents. 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-stranded and double-stranded nucleic acids. As used herein, the term "nucleic acid" also includes DNA as described above that contains 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 term "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refers 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 a combination thereof. The typical deoxyribonucleotides of DNA are thymine, adenine, cytosine, and guanine. The 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 a 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., one nucleotide) or polynucleotides (e.g., a sequence of nucleotides) relative to the base pairing rules. For example, the sequence 5'-AGT-3' is complementary to the sequence 3'-TCA-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 nucleic acids. The degree of complementarity between nucleic acid chains affects the efficiency and intensity of hybridization between nucleic acid chains. This is particularly important 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 comprising a coding sequence necessary to produce an RNA or polypeptide or its precursor. A functional polypeptide can be encoded by the full-length coding sequence or any portion of the coding sequence, as long as the desired activity or functional properties of the polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, etc.) are retained. When used to refer to a gene, the term "portion" refers to a fragment of the gene. The size of a fragment can range from a few nucleotides to the entire gene sequence minus one nucleotide. Therefore, "nucleotides comprising at least a portion of a gene" can include a fragment of a gene or the entire gene.

[0082] The term "gene" also includes the coding region of a structural gene and includes sequences located adjacent to the coding region at the 5' and 3' ends, for example, about 1 kb at either end, so that the gene corresponds to the length of the full-length mRNA (e.g., including coding, regulatory, structural, and other sequences). Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated or untranslated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated or 3' non-translated sequences. The term "gene" includes both cDNA and genomic forms of a gene. In some organisms (e.g., eukaryotes), the genomic form or clone of a gene contains a coding region interrupted by non-coding sequences called "introns" or "insertion regions" or "insertion sequences." Introns are gene segments that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are deleted or "spliced ​​out" from the nuclear or primary transcript and are therefore not present in the messenger RNA (mRNA) transcript. mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

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

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

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

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

[0087] "Amplification" is a special case of nucleic acid replication involving template specificity. It is contrasted with nonspecific template replication (i.e., replication that is template-dependent but not template-specific). Template specificity here is distinguished from the fidelity of replication (e.g., synthesis of the appropriate polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is often described in terms of "target" specificity. The target sequence is, in a sense, the "target" that is attempted to be selected from other nucleic acids. Amplification techniques are primarily designed for this selection.

[0088] Nucleic acid amplification 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 may be identical or non-identical), wherein the amplified product or amplicon is typically detectable. Polynucleotide amplification includes various chemical and enzymatic processes. Generating multiple DNA copies from one or several copies of a target or template DNA molecule during a polymerase chain reaction (PCR) or ligase chain reaction (LCR; see, e.g., U.S. Patent 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. Patent No. 5,639,611), assembly PCR (see, e.g., U.S. Patent No. 5,965,408), helicase-dependent amplification (see, e.g., U.S. Patent No. 7,662,594), hot-start PCR (see, e.g., U.S. Patent 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. Patent No. 5,508,169), methylation-specific PCR (see, e.g., Herman et al. (1996) PNAS 93(13):9821-9826), miniprimer 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, SA (2000) J. Molecular Endocrinology 25:169-193), solid phase PCR, thermal asymmetric staggered 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 fragments of a target sequence in a mixture of genomic DNA without cloning or purification. This method for amplifying a target sequence consists of introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycles in the presence of a DNA polymerase. The two primers are complementary to corresponding strands of the double-stranded target sequence. To perform amplification, the mixture is denatured, and the primers anneal to their complementary sequences within the target molecule. After annealing, the primers are amplified by a polymerase, forming a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated multiple times (i.e., denaturation, annealing, and extension constitute one "cycle"; there can be many "cycles") to obtain a high concentration of amplified fragments of the desired target sequence. The length of the amplified fragment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. Because of its repetitive nature, the process is called the "polymerase chain reaction" ("PCR"). Since the desired amplified segment of the target sequence becomes the predominant sequence (in terms of concentration) in the mixture, it is said to be "PCR amplified" and is a "PCR product" or "amplicon."

[0090] Template specificity is achieved by the selection of enzyme in most amplification techniques. Amplification enzyme is an enzyme that processes the specific sequence of nucleic acid only in the heterogeneous mixture of nucleic acid under the conditions of using them. For example, in the case of Q-β replicase, MDV-1RNA is the specific template of replicase (Kacian et al., Proc.Natl.Acad.Sci.USA, 69:3038

[1972] ). Other nucleic acids will not be copied by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has strict specificity (Chamberlin et al., Nature, 228:227

[1970] ) to its own promoter. In the case of T4 DNA ligase, enzyme will not connect two oligonucleotides or polynucleotides, wherein there is mismatch (Wu and Wallace (1989) Genomics 4:560) between the oligonucleotide or polynucleotide substrate and the ligation template at the junction. Finally, thermostable template-dependent DNA polymerases, such as Taq and Pfu DNA polymerases, were found to exhibit a high degree of specificity for primer binding, and thus defined, sequences due to their ability to function at elevated temperatures; elevated temperatures result in thermodynamic conditions that favor primer hybridization to the target sequence over non-target sequences (HA Erlich (ed.), PCR Technology, Stockton Press

[1989] ).

[0091] As used herein, the term "nucleic acid detection assay" refers to any method for 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 assays, Hologic, Inc.), and are described in, for example, 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); enzymatic 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); circulating probe technology (e.g., U.S. Patent Nos. 5,403,711, 5,011,769, and 5,660,988); Dade Behring signal amplification method (eg, 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 method (eg, 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 expected that the "amplifiable nucleic acid" will typically comprise a "sample template."

[0093] The term "sample template" refers to nucleic acids derived from a sample that are used to analyze the presence of a "target" (defined below). In contrast, "background template" is used to refer to nucleic acids other than the sample template, which may or may not be present in the sample. Background template is usually unintentional. This may be the result of legacy, or it may be due to the presence of nucleic acid contaminants that are attempted to be purified from the sample. For example, nucleic acids other than the nucleic acids to be detected from an organism may exist as background for the test sample.

[0094] The term "primer" refers to an oligonucleotide naturally occurring or synthetically produced in a restriction digest of purification, which can serve as a starting point for synthesis when induced to synthesize a primer extension product complementary to a nucleic acid chain (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH). The primer is preferably single-stranded for maximum efficiency in amplification, but may also be double-stranded. If double-stranded, the primer is first treated to separate its chain before being used to prepare an extension product. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to initiate the synthesis of an extension product in the presence of an inducing agent. The exact length of the primer will depend on many factors, including the use of temperature, primer source, and method.

[0095] The term "probe" refers to an oligonucleotide (e.g., a nucleotide sequence) naturally occurring in a purified restriction digest or synthesized, recombined, or produced by PCR amplification, which is capable of hybridizing with another target oligonucleotide. The probe can be single-stranded or double-stranded. The probe can be used for detection, identification, and separation of specific gene sequences (e.g., "capture probes"). It is expected that in some embodiments, any probe used in the present invention 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), fluorescence, radioactivity, and luminescence systems. The present invention is not intended to be limited to any specific detection system or label.

[0096] As used herein, "methylation" refers to methylation of cytosine at positions C5 or N4 of cytosine, N6 of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is typically unmethylated because typical in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" may also refer to amplified DNA that is unmethylated or methylated, respectively, from the original template.

[0097] Thus, as used herein, "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 recognized typical nucleotide bases. 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 the purposes of this article, thymine is not considered a methylated nucleotide when present in DNA, as thymine is a typical nucleotide base of DNA.

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

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

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

[0101] The methylation state of a nucleotide site in a nucleic acid molecule refers to the presence or absence of a methylated nucleotide at a specific site in the nucleic acid molecule. For example, when the nucleotide present at the 7th nucleotide in the nucleic acid molecule is 5-methylcytosine, the methylation state of the cytosine at the 7th nucleotide in the nucleic acid molecule is methylated. Similarly, when the nucleotide present at the 7th nucleotide in the nucleic acid molecule is cytosine (rather than 5-methylcytosine), the methylation state of the cytosine at the 7th nucleotide in the nucleic acid molecule is unmethylated.

[0102] The methylation state can optionally be represented or indicated by a "methylation value" (e.g., representing a methylation frequency, fraction, ratio, percentage, etc.). The methylation value 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 spectra after a bisulfite reaction, or by comparing the sequences of bisulfite-treated and untreated nucleic acids. Thus, a value such as a methylation value represents the methylation state and can therefore be used as a quantitative indicator of the methylation state in multiple copies of a locus. This is particularly useful when it is desired to compare the methylation state of a sequence in a sample with a threshold or reference value.

[0103] As used herein, "methylation frequency" or "percent (%) methylation" refers to the number of instances in which a molecule or locus is methylated relative to the number of instances in which the molecule or locus is not methylated.

[0104] Therefore, methylation state describes the methylation state of nucleic acid (for example, genomic sequence).In addition, methylation state refers to the characteristic relevant to methylation of the nucleic acid fragment at specific genomic site.These characteristics include but are not limited to whether any cytosine (C) residue in this DNA sequence is methylated, the position of methylated C residue, the frequency or percentage of methylated C in any specific region of nucleic acid, and the allelic difference in methylation, for example due to the difference of allelic origin. The terms "methylation state", "methylation signature" and "methylation profile" also refer to the relative concentration, absolute concentration or pattern of methylated C or unmethylated C in any particular region of a nucleic acid in a biological sample. For example, if the cytosine (C) residues in a nucleic acid sequence are methylated, it can be referred to as "hypermethylated" or as having "increased methylation", while if the sequence of cytosine (C) residues within a DNA sequence is not methylated, it can be referred to as "hypomethylated" or as having "decreased methylation". Similarly, if the cytosine (C) residues in a nucleic acid sequence are methylated compared to another nucleic acid sequence (e.g., from a different region or a different individual, etc.), the sequence is considered to be hypermethylated or as having increased methylation compared to the other nucleic acid sequence. Alternatively, if the cytosine (C) residues in a DNA sequence are not methylated compared to another nucleic acid sequence (e.g., from a different region or a different individual, etc.), the sequence is considered to be hypomethylated or as having decreased methylation compared to the other nucleic acid sequence. In addition, the present invention also provides a method for determining the relative concentration, absolute concentration or pattern of methylated C or unmethylated C in any particular region of a nucleic acid in a biological sample. The term "methylation pattern" used in the text refers to the collection site of methylated and unmethylated nucleotides on the nucleic acid region. When the number of methylated and unmethylated nucleotides is identical or similar in the entire region, but the position of methylated and unmethylated nucleotides is different, two nucleic acids may have identical or similar methylation frequencies or methylation percentages, but have different methylation patterns. When the degree of methylation of the sequence (for example, one has an increased or decreased methylation relative to another), frequency or pattern are different, the sequence is considered to be "differentially methylated" or to have a "methylation difference" or "different methylation states." The term "differential methylation" refers to the difference in nucleic acid methylation level or pattern in the cancer positive sample compared with the level or pattern of nucleic acid methylation in the cancer negative sample. It may also refer to the level or pattern difference between a patient with postoperative cancer recurrence and a patient without recurrence. The specific level or pattern of differential methylation and DNA methylation is a prognostic and predictive biomarker, for example, once the correct cut-off or prediction feature is determined.

[0105] Methylation state frequency can be used to describe the colony of individual or from single individual sample.For example, the nucleotide site with 50% methylation state frequency is methylated in 50% of the cases and unmethylated in 50% of the cases.Such frequency can be used to, for example, describe the degree of methylation of nucleotide site or nucleic acid region in the colony of individual or nucleic acid collection.Therefore, when the methylation in the first colony or nucleic acid molecule library is different from the methylation in the second colony or nucleic acid molecule library, the methylation state frequency of the first colony or library is different from the methylation state frequency of the second colony or library.Such frequency can also be used to, for example, describe the degree of methylation of nucleotide site or nucleic acid region in the single individual.For example, such frequency can be used to describe the degree of methylation of nucleotide site or nucleic acid region from the cell group of tissue sample or unmethylated.

[0106] As used herein, "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 a dinucleotide sequence comprising adjacent guanine and cytosine, wherein cytosine is located at the 5' (also referred to as CpG dinucleotide sequence) of guanine. In the human genome, most of the cytosines in CpG dinucleotides are methylated, yet some remain unmethylated in specific CpG dinucleotide-enriched genomic regions (referred to as CpG islands) (see, for example, Antequera et al. (1990) Cell 62:503–514).

[0108] As used herein, "CpG island" refers to a G:C-rich region of genomic DNA containing an increased number of CpG dinucleotides relative to the 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 the observed CpG frequency to the 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 the observed CpG frequency to the 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 × B) / (C × D), where R is the ratio of the observed CpG frequency to the expected frequency, A is the number of CpG dinucleotides in the analyzed sequence, B is the total number of nucleotides in the analyzed sequence, C is the total number of C nucleotides in the analyzed sequence, and D is the total number of G nucleotides in the analyzed sequence. Methylation status is often determined in CpG islands, such as in promoter regions. It will be appreciated, however, that other sequences in the human genome are also prone to DNA methylation, such as 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, the reagent or methylation-specific reagent that modifies the nucleotide of nucleic acid molecule as a function of the methylation state of nucleic acid molecule refers to a compound or composition or other reagent that can change the nucleotide sequence of nucleic acid molecule in a mode that reflects the methylation state of nucleic acid molecule. The method for treating nucleic acid molecule with such reagent can include contacting nucleic acid molecule with reagent, plus additional steps if necessary, to realize the desired change of nucleotide sequence. This change of the nucleotide sequence of nucleic acid molecule can result in a nucleic acid molecule in which each methylated nucleotide is modified into different nucleotides. This change of nucleic acid nucleotide sequence can result in a nucleic acid molecule in which each unmethylated nucleotide is modified into different nucleotides. This change of nucleic acid nucleotide sequence can result in a nucleic acid molecule in which each unmethylated selected nucleotide (for example, each unmethylated cytosine) is modified into different nucleotides. Using this reagent to change nucleic acid nucleotide sequence can result in a nucleic acid molecule in which each nucleotide (for example, each methylated cytosine) that is a methylated nucleotide is modified into different nucleotides. As used herein, a reagent that modifies a selected nucleotide is a reagent that modifies one of the four commonly 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 without modifying 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 unmethylated cytosine nucleotides. 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 methylated and unmethylated cytidines in, for example, CpG dinucleotide sequences.

[0111] The term "methylation assay" refers to any assay used to determine the methylation status 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 an art-recognized technique that allows global scanning of the genome using CG-rich primers to focus on regions most likely to contain CpG dinucleotides, as described by Gonzalgo et al. (1997) Cancer Research 57:594–599.

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

[0114] The term "Heavy Methyl TM ” refers to an assay in which a methylation-specific blocking probe (also referred to herein as a blocker) covering a CpG position located between or covered by an amplification primer is capable of methylation-specific selective amplification of a nucleic acid sample.

[0115] The term "Heavy Methyl TM MethyLight TM "Determination refers to HeavyMethyl TM MethyLight TM Assay, which is MethyLight TM Variations of the assay, including MethyLight TM The assay is combined with a methylation-specific blocking probe covering the CpG position between the amplification primers.

[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 US 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" (methylated 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 A1.

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

[0121] The term "methylation-specific restriction enzyme" or "methylation-sensitive restriction enzyme" refers to an enzyme that selectively digests nucleic acids based on the methylation state of its recognition site. In the case of a restriction enzyme that specifically cuts if the recognition site is not methylated or is methylated, if the recognition site is methylated, cutting does not occur or occurs with significantly reduced efficiency. In the case of a restriction enzyme that specifically cuts if the recognition site is methylated, if the recognition site is not methylated, cutting does not occur or occurs with significantly reduced efficiency. Preferably, a methylation-specific restriction enzyme has a recognition sequence containing a CG dinucleotide (e.g., a recognition sequence such as CGCG or CCCGGG). For some embodiments, it is further preferred that the restriction enzyme does not cut if the cytosine in the dinucleotide is methylated at carbon atom C5.

[0122] As used herein, " different nucleotides " refers to nucleotides that are chemically different from selected nucleotides, and different nucleotides are usually made to have Watson-Crick base pairing properties different from selected nucleotides, so that the commonly existing nucleotides complementary to selected nucleotides are different from the commonly existing nucleotides complementary to different nucleotides. For example, when C is the selected nucleotide, U or T can be different nucleotides, which are amplified by the complementation of C and G and the complementation of U or T and A. As used herein, the nucleotides complementary to selected nucleotides or complementary to different nucleotides refer to under high stringency conditions, with the nucleotides of selected nucleotides or different nucleotide base pairing, and its affinity is higher than the base pairing of three kinds of complementary nucleotides and four kinds of commonly existing nucleotides. The example of complementation is Watson-Crick base pairing in DNA (for example, AT and CG) and RNA (for example, AU and CG). Therefore, for example, under high stringency conditions, compared with G base pairing with G, A or T, G base pairs with C with higher affinity, and therefore, when C is the selected nucleotide, G is the nucleotide 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 for distinguishing between tumor and non-tumor samples. In some embodiments, a positive is defined as a histologically confirmed tumor reporting a DNA methylation value above a threshold value (e.g., a range associated with the disease), and a false negative is defined as a histologically confirmed tumor reporting a DNA methylation value below a threshold value (e.g., a range associated with the disease). Therefore, the value of sensitivity reflects the likelihood that the DNA methylation measurement of a given marker obtained from a known diseased sample is within the disease-related 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 that report DNA methylation values ​​below a threshold value for distinguishing between tumor and non-tumor samples. In some embodiments, a negative is defined as a histologically confirmed non-tumor sample that reports a DNA methylation value 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 that reports a DNA methylation value above a threshold value (e.g., a range associated with no disease). Thus, the specificity value reflects the likelihood that the DNA methylation measurement of a given marker obtained from a known non-tumor sample is within the non-disease-related measurement range. As defined herein, the clinical relevance of the 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 particularly refers to the area under the receiver operating characteristic (ROC) curve. The ROC curve is a graph of the true positive rate versus the false positive rate for different possible cut points for a diagnostic test. It shows the trade-off between sensitivity and specificity depending on the cut point chosen (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 will show an ROC curve with an area of ​​0.5 on the diagonal; Reference: JPEgan. (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, the growth of which exceeds and is out of proportion 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 that originates from a gland. Although these growths are benign, they may become worse over time.

[0128] The terms "precancer" or "preneoplastic" and their equivalents refer to any cell proliferative disorder that is undergoing malignant transformation.

[0129] A "site" or "region" of a tumor, adenoma, cancer, etc. is the tissue, organ, cell type, anatomical region, body part, etc., in a subject's body where 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 be exposed to a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to a treatment, determining the prognosis of a subject with a disease or condition (or its likely progression or regression), and determining the effect of a treatment on a subject with a disease or condition. For example, diagnostics can be used to detect the presence or likelihood that a subject is exposed to a tumor, or the likelihood that the subject will respond favorably to a compound (e.g., a drug, such as a pharmaceutical agent) or other treatment.

[0132] As used herein, the term "marker" refers to a substance (e.g., a nucleic acid or a nucleic acid region) that can diagnose a disease (e.g., a non-cancerous disease) (e.g., a cancerous disease) by distinguishing disease-associated cells (e.g., disease-associated non-cancerous cells) (disease-associated cancer cells) and normal cells, for example, based on methylation status.

[0133] When used to refer to nucleic acids, such as in "isolated oligonucleotides", the term "isolated" refers to a nucleic acid sequence that has been identified and separated from at least one contaminant nucleic acid with which it is normally associated in its natural source. An isolated nucleic acid exists in a form or setting different from that found in nature. In contrast, non-isolated nucleic acids, such as DNA and RNA, are found in a state in which they exist in nature. Examples of non-isolated nucleic acids include: a given DNA sequence (e.g., a gene) found on a host cell chromosome near adjacent genes; an RNA sequence, such as a specific mRNA sequence encoding a specific protein, which is found in a cell as a mixture with many other mRNAs encoding a large number of proteins. However, isolated nucleic acids encoding a specific protein include, by way of example, such nucleic acids in cells that normally express the protein, where the nucleic acid is in a chromosomal location different from that of the natural cell, or is flanked by nucleic acid sequences different from those found in nature. An isolated nucleic acid or oligonucleotide can exist 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 least the sense or coding strand (i.e., the oligonucleotide may be single-stranded), but may contain both sense and antisense strands (i.e., the oligonucleotide may be double-stranded). After separation from its natural or typical environment, the isolated nucleic acid can be combined with other nucleic acids or molecules.For example, the 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 has been 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 from other components with which it is naturally associated. As used herein, the term "purified" or "to purify" also refers to the removal of contaminants from a sample. Removal of contaminating proteins results in an increase in the percentage of the target polypeptide or nucleic acid 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 "composition comprising" a given polynucleotide sequence or polypeptide broadly refers to any composition comprising the given polynucleotide sequence or polypeptide. The composition may comprise an aqueous solution containing a salt (e.g., NaCl), a detergent (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 intended 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 include fluids, solids, tissues, and gases. Environmental samples include environmental materials, such as surface materials, soil, water, and industrial samples, etc. These examples should not be construed as limiting the sample types applicable to the present invention. In some embodiments, the sample includes esophageal tissue. In some embodiments, the sample includes esophageal tissue obtained by endoscopic brushing or using a non-endoscopic full esophageal brush or swabbing of a tethered device (such as a capsule sponge, balloon, or other device).

[0137] As used herein, a "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 a sample material originating from the pancreas is assessed in a stool sample (e.g., a sample that is 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 that undergoes 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 case of a reaction assay, this delivery system includes a system that allows reaction reagents (e.g., oligonucleotides, enzymes, etc. in a suitable container) and / or auxiliary materials (e.g., buffer, written instructions for measuring, etc.) to be stored, transported, or delivered from one location to another. For example, a test kit includes one or more housings (e.g., boxes) comprising relevant reaction reagents and / or auxiliary materials. As used herein, the term " fragmented kit " refers to a delivery system comprising two or more separate containers, each of which comprises a sub-portion of a total test kit component. The container can be delivered to an intended receiver together or separately. For example, a first container can comprise an enzyme for measuring, and a second container comprises oligonucleotides. The term " fragmented kit " is intended to include a test kit containing an analyte-specific reagent (ASR) as provided in Section 520 (e) of the Federal Food, Drug, and Cosmetic Act, but is not limited thereto. In fact, any delivery system comprising two or more separate containers, each of which comprises a sub-portion of a total test kit component, is included in the term " fragmented kit ". In contrast, a "combination kit" refers to a delivery system that includes all components of a reaction assay in a single container (eg, in a separate box containing each desired component). The term "kit" includes both separate and combination kits.

[0140] Implementation Methods of the Technology

[0141] Barrett's esophagus is the precursor lesion for most esophageal adenocarcinomas, malignancies with rapidly increasing incidence and persistently 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 may enable patients to enter surveillance programs, which may allow for detection of tumor progression at an earlier stage, enabling endoscopic or surgical treatment and improving outcomes. Screening for Barrett's esophagus and esophageal adenocarcinoma is limited by the lack of widely available tools and biomarkers that can be used in conjunction with screening tools. The acceptability and feasibility of screening by endoscopic and novel nonendoscopic methods have been demonstrated in populations. Nonendoscopic screening methods, such as swallowed cytology brushes or stool DNA testing, offer potentially cost-effective alternatives to endoscopy for identifying Barrett's esophagus in the general population. Recently, several abnormally methylated genes have been identified as highly discriminatory markers for Barrett's esophagus. In fact, a study of archived frozen esophageal biopsies from patients with and without Barrett's disease showed that a panel of tumor-associated genes could potentially be used to differentiate Barrett's esophagus from squamous mucosa (see, eg, Yang Wu, et al., DDW Abstract 2011).

[0142] Dysplasia is known to be distributed in a patchy manner in Barrett's esophagus, resulting in "sampling error" of conventional endoscopic surveillance with four-quadrant biopsies. Conventional endoscopic surveillance is known to utilize biopsy samples of less than 10% of the BE segment. Compliance among endoscopists utilizing conventional surveillance is known to be poor. Although newer endoscopic technologies have been shown to increase the yield of dysplasia detection in studies conducted at tertiary care centers, their applicability in the population remains uncertain. Methods of sampling larger mucosal surface areas (such as swabs or brushes) may increase the yield of dysplasia and tumors, especially if combined with molecular markers of dysplasia / tumors. This may ultimately allow non-biopsy (by swab or brush) or non-endoscopic surveillance of BE subjects with substantial potential cost savings.

[0143] Thus, provided herein are techniques for esophageal disease screening, particularly, but not exclusively, methods, compositions, and related uses for detecting the presence of esophageal diseases, such as Barrett's esophagus, Barrett's esophagus dysplasia, and the like. Furthermore, the present technology provides methods, compositions, and related uses for distinguishing between Barrett's esophagus and Barrett's esophagus dysplasia, as well as low-grade dysplasia, high-grade dysplasia, and esophageal adenocarcinoma in samples obtained by endoscopic brushing or non-endoscopic whole esophageal brushing or swabbing using a tethered device, such as a capsule sponge, balloon, or other device.

[0144] In fact, experiments conducted during the development of the present technology compared the methylation status of DNA markers from esophageal tissue of subjects with Barrett's esophagus with the methylation status of the same DNA markers from control subjects (e.g., normal tissue of each tissue type) and with the methylation status of the same DNA markers from subjects with Barrett's esophagus dysplasia (see Examples 1-4).

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

[0146] Markers and / or marker panels (eg, chromosomal regions with annotations provided in Tables 2, 3, 5, and / or 6) capable of classifying BE and Barrett's esophagus-related dysplasia (BED) in esophageal tissue were identified (see Examples 1, 3, and 4).

[0147] Markers and / or marker panels (e.g., chromosomal regions with annotations provided in Table 5) were identified that are capable of predicting Barrett's esophagus-associated low-grade dysplasia (BE-LGD), Barrett's esophagus-associated dysplasia-high-grade dysplasia (BE-HGD), and esophageal adenocarcinoma (EAC) in samples obtained by whole esophageal swab or brushing (see Examples 1 and 4).

[0148] Markers and / or marker panels (eg, chromosomal regions with annotations provided in Table 5) were identified that are capable of classifying BE and BED in samples obtained by whole esophageal swab or brushing (see Examples 1 and 4).

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

[0150] The method comprises determining the methylation state of at least one methylation marker in a biopsy sample isolated from a subject, wherein a change in the methylation state of the marker indicates the presence or type of esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC). Specific embodiments relate to markers comprising differentially methylated regions (DMRs, e.g., DMRs 1-229, see Tables 1, 2, 3, 5, 6, 7, 8) for diagnosing (e.g., screening) esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC), including early detection in precancerous stages of the disease (e.g., BE versus BED).

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

[0152] In addition to embodiments in which methylation analysis is performed for at least one marker, region of a marker, or base of a marker comprising 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 a marker panel in esophageal tissue comprising at least one marker, region of a marker, or base of a marker containing a DMR for detecting esophageal diseases (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 comprising a DMR.

[0154] In some embodiments, the present technology provides for determining the methylation state 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., DMR1-229)) using a combination of bisulfite technology and one or more methylation assays. Genomic CpG dinucleotides can be methylated or unmethylated (also referred to as upmethylated and downmethylated, respectively). However, the methods of the present invention are suitable for analyzing biological samples of a heterogeneous nature, such as low concentrations of tumor cells in the background of remote samples (e.g., blood, organ effluent, or feces), or biological material therefrom. Therefore, when analyzing the methylation state of a CpG position in such a sample, a quantitative assay can be used to determine the methylation level (e.g., percentage, fraction, ratio, proportion, or degree) 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 (eg, BE, BED, BE-LGD, BE-HGD, EAC).

[0156] Combination of markers

[0157] In some embodiments, the present technology involves assessing the methylation state of a marker combination 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 state of more than one marker increases the specificity and / or sensitivity of screening or diagnosis for identifying esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC) in a subject.

[0158] Various cancers are predicted by various combinations of markers, such as those identified by statistical techniques that correlate specificity and sensitivity of the predictions. The present technology provides methods for identifying predictive combinations for certain cancers and validating predictive combinations.

[0159] In some embodiments, a combination of markers (eg, comprising a DMR) predicts the location of a tumor.

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

[0161] Markers and / or marker panels (eg, chromosomal regions with annotations provided in Tables 2, 3, 5, and / or 6) capable of classifying BE and Barrett's esophagus-related dysplasia (BED) in esophageal tissue were identified (see Examples 1, 3, and 4).

[0162] Markers and / or marker panels (e.g., chromosomal regions with annotations provided in Table 5) were identified that are capable of predicting Barrett's esophagus-associated low-grade dysplasia (BE-LGD), Barrett's esophagus-associated dysplasia-high-grade dysplasia (BE-HGD), and esophageal adenocarcinoma (EAC) in samples obtained by whole esophageal swab or brushing (see Examples 1 and 4).

[0163] Markers and / or marker panels (eg, chromosomal regions with annotations provided in Table 5) were identified that are capable of classifying BE and BED in samples obtained by whole esophageal swab or brushing (see Examples 1 and 4).

[0164] Methods used to analyze methylation status

[0165] The most commonly used method for analyzing nucleic acids for the presence of 5-methylcytosine is based on the bisulfite method described by Frommer et al. (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827–31) or its variations. The bisulfite method for detecting 5-methylcytosine in DNA is based on the observation that cytosine, rather than 5-methylcytosine, reacts with bisulfite ions (also called bisulfite). The reaction generally proceeds as follows: First, cytosine reacts with bisulfite to form sulfonated cytosine. Next, the sulfonated intermediate undergoes spontaneous deamination to yield sulfonated uracil. Finally, the sulfonated uracil is desulfonated under alkaline conditions to form uracil. Detection is possible because uracil forms base pairs with adenine (thus behaving like thymine), whereas 5-methylcytosine forms base pairs with guanine (thus behaving like cytosine). This allows differentiation of methylated from unmethylated cytosines 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 blocking the DNA to be analyzed in an agarose matrix to prevent DNA diffusion and renaturation (bisulfite reacts only with single-stranded DNA) and replacing 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). This allows analysis of the methylation status of individual cells, demonstrating the practicality 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 typically involve amplifying a short, specific fragment of a known nucleic acid after bisulfite treatment, followed 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. Pat. 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 the bisulfite technique for methylation detection of single 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 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 the use of methylation-sensitive restriction enzymes.

[0169] For example, genomic sequencing has been simplified to analyze methylation patterns and 5-methylcytosine distribution by using bisulfite treatment (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, by Sadri & Hornsby (1997) Nucl. Acids Res. 24: 5058–5059, or as embodied by the method known as COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird (1997) Nucleic Acids Res. 25: 2532–2534).

[0170] COBRA TM Analysis is a quantitative methylation assay for determining the level of DNA methylation at a specific locus 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 the methylation-dependent sequence differences in the PCR products of sodium 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). PCR amplification of the DNA converted by bisulfite is then performed using primers specific for the target CpG island, followed by restriction endonuclease digestion, gel electrophoresis, and detection using a specifically labeled hybridization probe. The methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR products in a linear quantitative manner over the entire broad spectrum of DNA methylation levels. Furthermore, the present technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.

[0171] For COBRA TM Typical reagents for the assay (e.g., can be found in a typical COBRA-based TM The kits (found in the kits) 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. In addition, bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffer and DNA recovery components.

[0172] Preferably, such as "MethyLight TM "(Real-time PCR technology based on fluorescence) (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 methylated CpG island amplification ("MCA"; Toyota et al., Cancer Res. 59:2307-12, 1999) are used alone or in combination with one or more of these methods.

[0173] “Heavy Methyl TM The assay is a quantitative method for assessing methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific blocking probes ("blockers") covering CpG positions located between or covered by amplification primers enable methylation-specific selective amplification of nucleic acid samples.

[0174] The term "Heavy Methyl TM MethyLight TM "Determination refers to HeavyMethyl TM MethyLight TM Assay, which is MethyLight TM Variations of the assay, including MethyLight TM The assay is combined with a methylation-specific blocking probe covering the CpG position between the amplification primers. TM The assay can also be used in combination with methylation-specific amplification primers.

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

[0176] MSP (methylation-specific PCR) allows the assessment of the methylation status of virtually any group of CpG sites 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; U.S. Patent No. 5,786,146). Briefly, DNA is modified with sodium bisulfite, which converts unmethylated cytosines, but not methylated cytosines, to uracil, and the product is subsequently amplified using primers specific for both methylated and unmethylated DNA. MSP requires only small amounts 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 for MSP analysis (e.g., as found in a typical MSP-based kit) can 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 is performed using fluorescence-based real-time PCR (e.g. ) is a high-throughput quantitative methylation assay that requires no further manipulation after the PCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). 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 standard procedures (bisulfite method to convert 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 the level of the amplification process and the level of the fluorescence detection process.

[0178] MethyLight TM The assay is used as a quantitative test of methylation patterns in nucleic acids such as genomic DNA samples, where sequence discrimination occurs at the probe hybridization level. In the quantitative version, a PCR reaction provides methylation-specific amplification in the presence of a fluorescent probe that overlaps a specific putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers nor the probe cover any CpG dinucleotides. Alternatively, the amount of methylation can be determined by using a control oligonucleotide (e.g., HeavyMethyl) that does not cover a known methylation site. TMQualitative testing of genomic methylation can be achieved by using PCR (fluorescence-based versions of the genomic methylation and MSP technologies) or by probing biased PCR libraries with oligonucleotides covering potential methylation sites.

[0179] MethyLight TM Methods and any suitable probes (e.g. probe, For example, in some applications, double-stranded genomic DNA is treated with sodium bisulfite and subjected to probes, such as using MSP primers and / or HeavyMethyl blocker oligonucleotides and One of two PCR reactions for the probe. The probe is dual-labeled with a fluorescent "reporter" and a "quencher" molecule and is designed to be specific for regions of relatively high GC content, so that it melts during PCR cycles at a temperature approximately 10°C higher than that of the forward or reverse primer. The probe remains fully hybridized during the PCR annealing / extension step. As Taq polymerase enzymatically synthesizes new strands during PCR, it eventually reaches the annealed probe. The Taq polymerase 5' to 3' endonuclease activity will digest The probe releases a fluorescent reporter molecule to replace probes to quantitatively detect their now-unextinguished signals using a real-time fluorescence detection system.

[0180] Used for MethyLight TM Typical reagents for the assay (e.g., can be found in typical MethyLight-based TM ) can 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.); or probe; optimized PCR buffer and deoxynucleotides; and Taq polymerase.

[0181] QM TM (Quantitative methylation) assay is an alternative quantitative test for methylation patterns in genomic DNA samples, in which sequence discrimination occurs at the probe hybridization level. In this quantitative version, the PCR reaction provides unbiased amplification in the presence of a fluorescent probe that overlaps with a specific putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers nor the probe cover any CpG dinucleotides. Alternatively, the amount of input DNA is determined by using a control oligonucleotide (e.g., HeavyMethyl) that does not cover a known methylation site. TMQualitative testing of genomic methylation can be achieved by using PCR (fluorescence-based versions of the genomic methylation and MSP technologies) or by probing biased PCR libraries with oligonucleotides covering potential methylation sites.

[0182] During the amplification process, QM TM The method can be used with any suitable probe (e.g. probe, For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and probe. The probe is dual-labeled with a fluorescent "reporter" and a "quencher" molecule and is designed to be specific for regions of relatively high GC content, so that it melts during PCR cycles at a temperature approximately 10°C higher than that of the forward or reverse primer. The probe remains fully hybridized during the PCR annealing / extension step. As Taq polymerase enzymatically synthesizes new strands during PCR, it eventually reaches the annealed probe. The Taq polymerase 5' to 3' endonuclease activity will digest The probe releases a fluorescent reporter molecule to replace probes to quantitatively detect their current undying signals using a real-time fluorescence detection system. TM Typical reagents for the analysis (e.g., can be found in typical QM-based TM ) can 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.); or probe; optimized PCR buffer and deoxynucleotides; and Taq polymerase.

[0183] Ms-SNuPE TM The technology is a quantitative method based on bisulfite treatment of DNA followed by single nucleotide primer extension to assess methylation differences at specific CpG sites (Gonzalgo & Jones, Nucleic Acids Res. 25: 2529-2531, 1997). In brief, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine into uracil, while keeping 5-methylcytosine unchanged. PCR primers specific for bisulfite-converted DNA are then used to amplify the desired target sequence, and the resulting product is separated and used as a template for methylation analysis at the target CpG site. A small amount 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 the analysis (e.g., can be found in typical Ms-SNuPE-based TM The kits (found in the kits) 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; Ms-SNuPE for specific loci; TM Primers; reaction buffer (for Ms-SNuPE reaction); and labeled nucleotides. In addition, bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery reagents or kits (such as precipitation, ultrafiltration, affinity column); desulfonation buffer and DNA recovery components.

[0185] Reduced Representation Bisulfite Sequencing (RRBS) begins with bisulfite treatment of nucleic acids to convert all unmethylated cytosines into uracil, followed by restriction enzyme digestion (e.g., by enzymes such as MspI that recognize sites containing CG sequences) and complete sequencing of the fragments after coupling to adapter ligands. The selection of restriction enzymes enriches fragments in CpG-dense regions, reducing the number of redundant sequences that may be mapped to multiple gene positions during the analysis process. 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 a high frequency of restriction enzyme cleavage sites in these regions, thereby providing a determination of the methylation status of one or more genomic loci.

[0186] A typical protocol for RRBS includes digestion of nucleic acid samples with a restriction enzyme such as Mspl, filling in overhangs and A-tails, ligating adapters, bisulfite conversion, and PCR. See, for example, Meissner 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, methylation status is assessed using quantitative allele-specific real-time target and signal amplification (QuARTS) assays. Three reactions occur sequentially in each QuARTS assay, including amplification (reaction 1) and target probe cleavage (reaction 2) in the primary reaction; and FRET cleavage and fluorescence signal generation (reaction 3) in the secondary reaction. When the target nucleic acid is amplified with specific primers, a specific detection probe with a flanking sequence loosely binds to the amplicon. The presence of a specific invasive oligonucleotide on the target binding site causes the cleavage enzyme to release the flanking sequence by cutting between the detection probe and the flanking sequence. The flanking sequence is complementary to the non-hairpin portion of the corresponding FRET box. Therefore, the flanking sequence acts as an invasive oligonucleotide on the FRET box and achieves cleavage between the FRET box fluorophore and the quencher to generate a fluorescent signal. The cleavage reaction can cut multiple probes per target, so each flank releases multiple fluorophores, providing exponential signal amplification. QuARTS can detect multiple targets in a single reaction well by using FRET boxes 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 carried out in the presence of a denaturing solvent such as, but not limited to, n-alkyl glycol or diglyme (DME) or in the presence of dioxane or a dioxane derivative. In some embodiments, the denaturing solvent is used at a concentration of 1% to 35% (v / v). In some embodiments, the bisulfite reaction is carried out in the presence of a scavenger such as, but not limited to, a chroman derivative such as 6-hydroxy-2,5,7,8,-tetramethylchroman 2-carboxylic acid or trihydroxybenzoic acid and its derivatives such as gallic acid (see PCT / EP2004 / 011715). Preferably, the bisulfite conversion is carried out at a reaction temperature of 30°C to 70°C, whereby the temperature is briefly raised to above 85°C during the reaction (see PCT / EP2004 / 011715). The bisulfite-treated DNA is preferably purified prior to quantification. This can be done by any means known in the art, such as, but not limited to, ultrafiltration, e.g., by Microcon TM Column (Millipore TM Purification was performed according to a modified manufacturer's protocol (see, for example, PCT / EP2004 / 011715).

[0189] In some embodiments, use according to primer oligonucleotide groups of the present invention (for example, referring to Tables 4 and 9) and amplification enzyme to increase the fragment of processed DNA. The amplification of multiple DNA fragments can be carried out simultaneously in the same reaction vessel. Usually, polymerase chain reaction (PCR) is used to increase. The length of amplicon is generally 100 to 2000 base pairs.

[0190] In another embodiment of the method, the methylation status of a CpG position within or near a marker comprising a DMR (e.g., DMR1-229 provided in Tables 1, 2, 3, 5, 6, 7, 8) can be detected using methylation-specific primer oligonucleotides. This technology (MSP) has been described in U.S. Pat. No. 6,265,171 to Herman. Amplification of bisulfite-treated DNA using methylation-state-specific primers allows for the distinction between methylated and unmethylated nucleic acids. The MSP primer pair contains at least one primer that hybridizes to a bisulfite-treated CpG dinucleotide. Thus, the sequence of the primer contains at least one CpG dinucleotide. The MSP primer that is specific for unmethylated DNA contains a "T" at the position of the C position in the CpG.

[0191] The fragment obtained by amplification can carry direct or indirect detectable labeling.In some embodiments, labeling is a fluorescent marker, a radionuclide or a separable molecular fragment with a typical mass that can be detected in a mass spectrometer.When the labeling is a mass marker, some embodiments provide the amplicon with the labeling of a single positive or negative net charge, thereby allowing to have better detectability in a mass spectrometer.Can be detected and visualized by for example matrix-assisted laser desorption / ionization mass spectrometry (MALDI) or use electrospray ionization mass spectrometry (ESI).

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

[0193] method

[0194] In some embodiments of the present technology, a method is provided comprising the steps of:

[0195] 1) contacting a nucleic acid obtained from a subject (e.g., genomic DNA, e.g., isolated from a body fluid such as a stool sample, a blood sample, or a tissue sample (e.g., esophageal tissue)) with at least one reagent or a series of reagents that distinguishes between methylated and unmethylated CpG dinucleotides in at least one marker, the 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 (eg, 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, a method is provided comprising the steps of:

[0198] 1) contacting a nucleic acid obtained from a subject (e.g., genomic DNA, e.g., isolated from a body fluid such as a stool sample, a blood sample, or a tissue sample (e.g., esophageal tissue)) with at least one reagent or a series of reagents that distinguishes between methylated and unmethylated CpG dinucleotides in at least one marker, the 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 (eg, 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, a method is provided comprising the steps of:

[0201] 1) contacting a nucleic acid obtained from a subject (e.g., genomic DNA, e.g., isolated from a body fluid such as a stool sample, a blood sample, or a tissue sample (e.g., esophageal tissue)) with at least one reagent or a series of reagents that distinguishes between methylated and unmethylated CpG dinucleotides in at least one marker, the at least one marker comprising a DMR (e.g., DMR Nos. 3, 5, 30, 33, 43, 58, 77, and 79-128 provided in Table 2; DMR Nos. 77, 27, 193, 90, 92, 101, and 129-134 provided in Table 3; DMR Nos. 77, 90, and 135 provided in Table 5; and DMR Nos. 136-187 provided in Table 6) and

[0202] 2) Classification as Barrett's esophagus or Barrett's esophagus dysplasia (eg, 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, a method is provided comprising the steps of:

[0204] 1) contacting nucleic acid obtained from a subject (e.g., genomic DNA, e.g., isolated from esophageal tissue (e.g., esophageal tissue obtained by whole esophageal swabbing or brushing)) with at least one reagent or a series of reagents that distinguish between methylated and unmethylated CpG dinucleotides in at least one marker, the at least one marker comprising a DMR (e.g., DMR Nos. 77, 90, and 135 provided in Table 5) and

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

[0206] In some embodiments of the present technology, a method is provided comprising the steps of:

[0207] 1) contacting nucleic acid obtained from a subject (e.g., genomic DNA, e.g., isolated from esophageal tissue (e.g., esophageal tissue obtained by whole esophageal swabbing or brushing)) with at least one reagent or a series of reagents that distinguish between methylated and unmethylated CpG dinucleotides in at least one marker, the at least one marker comprising a DMR (e.g., DMR Nos. 77, 90, and 135 provided in Table 5) and

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

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

[0210] Genomic DNA can be isolated by any means, including the use of commercially available test kits. In short, where the target DNA is encapsulated by the cell membrane, the biological sample must be destroyed and cracked by enzymes, chemicals, 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 the solution. This can be done by a variety of methods, including salting out, organic extraction, or combining the DNA with a solid phase carrier. The choice of method is affected by multiple factors, including time, cost, and the amount of DNA required. All clinical sample types containing tumor material or pre-tumor material are suitable for this method, such as cell lines, histological sections, biopsies, paraffin-embedded tissues, body fluids, feces, colon effluent, urine, plasma, serum, whole blood, separated blood cells, cells separated 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 whole esophageal brushing or swabbing using a tethered device (eg, a capsule sponge, balloon, or other device).

[0213] The present technology is not limited to the method for preparing samples and providing nucleic acids for testing. For example, in some embodiments, DNA is isolated from stool samples or from blood or from plasma samples using direct gene capture (e.g., as described in U.S. Patent Application Serial No. 61 / 485,386) or related methods.

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

[0215] In some embodiments, the reagent converts a cytosine base that is unmethylated at the 5'-position to uracil, thymine, or another base that is dissimilar to cytosine in hybridization behavior. However, in some embodiments, the reagent may be a methylation-sensitive restriction enzyme.

[0216] In some embodiments, the genomic DNA sample is treated in such a way that the unmethylated cytosine base at the 5'-position is converted to uracil, thymine, or another base that is dissimilar to cytosine in hybridization behavior. In some embodiments, this treatment is performed with a bisulfate (bisulfite, disulfite) followed by alkaline hydrolysis.

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

[0218] The present technology relates to the analysis of any sample relevant to esophageal disease (such as BE, BED, BE-LGD, BE-HGD, EAC). For example, in some embodiments, the sample comprises tissue and / or biological fluid obtained from the patient. In some embodiments, the sample comprises esophageal tissue. In some embodiments, the sample comprises esophageal tissue obtained by wiping or brushing the whole esophagus. In some embodiments, the sample comprises secretions. In some embodiments, the sample comprises blood, serum, plasma, gastric secretions, pancreatic juice, microdissected cells of gastrointestinal biopsy samples, esophageal biopsy, esophageal cells shed into the gastrointestinal cavity and / or esophageal cells recovered from feces. 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 gastrointestinal tract and the 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 cell fluid, ascites, urine, feces, pancreatic juice, fluid obtained during endoscopy, blood, mucus, or saliva. In some embodiments, the sample is a fecal sample.

[0219] In some embodiments, the present invention provides the method for the preparation of the present invention.Such sample can be obtained by any number of methods known in the art, such as, those that are apparent to those skilled in the art.For example, urine and fecal samples are easily obtained, and blood, ascites, serum or pancreatic juice samples can be obtained, for example, by using a needle and syringe parenteral.Acellular or substantially acellular samples can be obtained by subjecting the sample to various techniques known to those skilled in the art (including but not limited to centrifugation and filtration).Although it is usually preferred not to use invasive techniques to obtain sample, it may still be preferred to obtain samples such as tissue homogenate, tissue section and biopsy specimens.In some embodiments, the sample is obtained by swabbing or brushing or using a sponge capsule device to swab the esophagus.

[0220] In some embodiments, the present technology relates to methods of treating a patient (e.g., a patient with BE, BED, BE-LGD, BE-HGD, and / or EAC), 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 surgery, imaging the patient, or 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 patients 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 for diagnosing an esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC) in a subject are provided. As used herein, the terms "performing a diagnosis" and "diagnosing" refer to methods by which one skilled 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 skilled 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 state of which indicates the presence, severity, or absence of a condition.

[0222] Following 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 an assessment of the potential risk of developing cancer can be made, appropriate treatment can be selected, and in some cases, a less severe treatment can be chosen for the patient. Assessment of cancer biomarkers (e.g., determining methylation status) can be used to separate subjects with a good prognosis and / or a low risk of developing cancer who will not require treatment or will require limited treatment, from subjects who are more likely to develop cancer or suffer a recurrence of cancer and will benefit from more intensive treatment.

[0223] Thus, "making a diagnosis" or "diagnosis" as used herein 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 current treatment and potentially changing treatment based on measurements of the diagnostic biomarkers (e.g., DMRs) provided herein. In addition, in some embodiments of the subject matter of the present disclosure, multiple measurements of biomarkers over time can be performed to aid in diagnosis and / or prognosis. Temporal changes in biomarkers can be used to predict clinical outcome, monitor the progression of esophageal disease, and / or monitor the effectiveness of appropriate treatments for cancer. In such embodiments, for example, over the course of effective treatment, it is expected that changes in the methylation state of one or more biomarkers disclosed herein (e.g., DMRs) (and potentially one or more additional biomarkers, if monitored) in a biological sample can be seen over time.

[0224] The subject matter of the present disclosure further provides, in some embodiments, a method for determining whether to initiate or continue prevention or treatment of an esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC) in a subject. In some embodiments, the method comprises providing a series of biological samples from a subject over time; analyzing the series of biological samples to determine the methylation state of at least one biomarker disclosed herein in each biological sample; and comparing any measurable changes in the methylation state of one or more biomarkers in each biological sample. Any change in the methylation state of the biomarker during this period 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 before the start of treatment, and a second time point can be selected at some time after the start of treatment. The methylation state can be measured in each sample taken from different time points, and qualitative and / or quantitative differences can be recorded. Changes in the methylation state of the biomarker levels from different samples can be correlated with the subject's esophageal disease risk, prognosis, determination of treatment efficacy, and / or progression of esophageal disease.

[0225] In preferred embodiments, the methods and compositions of the present invention are used to treat or diagnose a disease at an early stage, for example, before symptoms of the disease appear. In some embodiments, the methods and compositions of the present invention are used to treat or diagnose a disease at a clinical stage.

[0226] As described above, in some embodiments, multiple determinations of one or more diagnostic or prognostic biomarkers can be performed, and the time variation of the marker can be used to determine diagnosis or prognosis. For example, a diagnostic marker can be determined at the initial time, and the diagnostic marker can be determined again at the second time. In such an embodiment, the increase in the marker from the initial time to the second time can diagnose a specific type or severity of esophageal disease, or a given prognosis. Similarly, the reduction in the marker from the initial time to the second time can indicate a specific type or severity of esophageal disease, or a given prognosis. In addition, the degree of change of one or more markers can be relevant to the severity of esophageal disease and future adverse events. It will be understood by those skilled in the art that, although in certain embodiments, the same biomarker can be compared and measured at multiple time points, a given biomarker can also be measured at one time point, and a second biomarker can be measured at the second time point, and the comparison of these markers can provide diagnostic information.

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

[0228] In some embodiments, statistical analysis is associated with the tendency of prognostic indicators and unfavorable results.For example, in some embodiments, the methylation state different from the methylation state in the normal control sample obtained from the patient without esophageal disease can show that, compared with the subject with the level more similar to the methylation state in the control sample, the subject is more likely to suffer from esophageal disease, as determined by statistical significance level. In addition, the change of methylation state from baseline (such as "normal") level can reflect the prognosis of the subject, and the degree of change of methylation state can be related to the severity of adverse events. Usually by comparing two or more colonies and determining confidence interval and / or p value to determine statistical significance (see, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983). Exemplary confidence intervals for the present subject matter 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 value variation of the methylation state of a prognostic or diagnostic biomarker (e.g., DMR) disclosed herein can be established, and the variation of the methylation state of the biomarker in the biological sample is simply compared with the threshold value variation of the methylation state. The preferred threshold value variation of the methylation state of the biomarker provided herein is 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 state of a prognostic or diagnostic indicator (a combination of biomarkers or biomarkers) is directly correlated with the associated tendency of a given result. Those skilled in the art are familiar with using such a nomogram to associate two numerical values, and recognize that the uncertainty of the measurement is the same as the uncertainty of the marker concentration because a single sample measurement is cited, rather than a population average.

[0230] In some embodiments, a control sample is analyzed simultaneously with the biological sample so that the results obtained from the biological sample can be compared with the results obtained from the control sample. In addition, it is expected that a standard curve can be provided, using which the measurement results of the biological sample can be compared. If a fluorescent label is used, such a standard curve presents the methylation state of the biomarker as a function of the measurement unit (e.g., fluorescence signal intensity). Using samples obtained from multiple donors, a standard curve of the "risk level" of one or more biomarkers in normal tissue can be provided, as well as a standard curve of the "risk level" of one or more biomarkers in tissue obtained 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 the method, when an abnormal methylation state of one or more DMRs provided herein is identified in a biological sample obtained from a subject, the subject is identified as having esophageal disease. In other embodiments of the method, the abnormal methylation state of one or more such biomarkers detected in a biological sample obtained from a subject causes the subject to be identified as having esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC).

[0231] The analysis of marker can be carried out separately in a test sample or carried out simultaneously with other marker.For example, multiple marker combination can be used for effectively processing multiple samples and potentially provide better diagnosis and / or prognosis accuracy in a test.In addition, those skilled in the art will recognize that test is from the value of multiple samples (for example, at continuous time point) of same experimenter.Testing a series of samples like this can allow to identify the variation of marker methylation state over time.The variation of methylation state and the absence of the variation of methylation state can provide useful information about morbid state, include but not limited to determine the approximate time that event starts, the existence and amount of curable tissue, the suitability of drug therapy, the effectiveness of various therapies, identify the result of experimenter, comprise the risk of future event.

[0232] Biomarker analysis can be performed in a variety of physical formats. For example, microtiter plates or automation can be used to facilitate the processing of large numbers of test samples. Alternatively, single-sample formats can be developed to facilitate prompt, point-of-care treatment and diagnosis, such as 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 there is a measurable change in the methylation state of at least one biomarker in the sample compared to the control methylation state. Conversely, when no change in methylation state is identified in the biological sample, the subject can be identified as not having the esophageal disease, not having a risk of the esophageal disease, or having a low risk of the esophageal disease. In this regard, subjects with esophageal disease or its risk can be distinguished from subjects with low to substantially no esophageal disease or its risk. Subjects at risk of developing esophageal disease can be placed on a more intensive and / or regular screening regimen, including endoscopic monitoring. On the other hand, subjects with low to substantially no risk can avoid endoscopy or esophageal brushing until a future screening time, such as when screening performed according to the present technology indicates that a risk of esophageal disease has developed in these subjects.

[0234] As described above, according to embodiments of the methods of the present technology, detecting a change in the methylation state of one or more biomarkers can be a qualitative determination or a quantitative determination. Thus, the step of diagnosing a subject as having an esophageal disease or being at risk for developing an esophageal disease indicates making certain threshold measurements, for example, a change in the methylation state of one or more biomarkers in a biological sample compared to a predetermined control methylation state. In some embodiments of the method, the control methylation state is any detectable methylation state of the biomarker. In other embodiments of the method in which a control sample is tested simultaneously with the biological sample, the predetermined methylation state is the methylation state in the control sample. In other embodiments of the method, the predetermined methylation state is based on and / or identified by a standard curve. In other embodiments of the method, the predetermined methylation state is a specific state or range of states. Thus, the predetermined methylation state can be selected within limits apparent to one skilled in the art, based in part on the embodiment of the method being implemented and the desired specificity, among other things.

[0235] Furthermore, 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 both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. Thus, the present technology provides for the diagnosis of mammals, such as humans, as well as mammals that are important due to their endangerment, such as Siberian tigers; economically important mammals, such as animals raised on farms for human consumption; and / or animals that are socially important to humans, such as animals kept as pets or in zoos. Examples of such animals include, but are not limited to: carnivores such as cats and dogs; suids, including pigs, hogs, and wild boars; ruminants and / or ungulates, such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Thus, the diagnosis and treatment of livestock, including, but not limited to, domesticated pigs, ruminants, ungulates, horses (including racehorses), and the like, are also provided. The subject matter of the present disclosure also includes systems for diagnosing esophageal disease (e.g., BE, BED, BE-LGD, BE-HGD, EAC) in a subject. The system can be provided, for example, as a commercial kit that can be used to screen for risk of esophageal disease in a subject from whom a biological sample has been collected or to diagnose esophageal disease. Exemplary systems provided according to the present technology include assessing the methylation status of DMRs, such as the DMRs provided in Tables 1, 2, 3, 5, 6, 7, and / or 8. DETAILED DESCRIPTION

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

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

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

[0239] b) 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 Barrett's esophagus dysplasia, wherein the marker comprises a base in a differentially methylated region (DMR) selected from BMP3, NDRG4, VAV3, SFMBT2, DIO3, HUNK, ELMO1, 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 whole esophageal swabbing or brushing or using a sponge capsule device.

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

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

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

[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 assay utilizes a methylation-specific nuclease.

[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, wherein the marker comprises a base in a DMR selected from the group consisting of BMP3, NDRG4, VAV3, SFMBT2, DIO3, HUNK, ELMO1, CD1D, CDKN2A, and OPLAH;

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

[0253] c) determining a confidence interval and / or p-value for the difference in methylation status between 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 whole 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, ELMO1, 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; and comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to the nucleotide sequence of a nucleic acid containing the DMR from:

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

[0259] b) subjects without Barrett's esophagus dysplasia, to identify differences in the two sequences; and

[0260] When the difference in a) and b) exists, 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 whole 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 the sample; a software component configured to compare the methylation status of the sample with the methylation status of a control sample or reference sample recorded in a database; and an alarm component configured to determine a single value based on a combination of methylation states and alert a user of a methylation status associated with Barrett's esophagus.

[0264] 20. The system of embodiment 19, wherein the sample comprises a nucleic acid comprising a DMR selected from the group consisting of BMP3, NDRG4, VAV3, SFMBT2, DIO3, HUNK, ELMO1, 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 with Barrett's esophagus, from subjects without Barrett's esophagus, and from subjects without Barrett's esophagus dysplasia.

[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 an agent 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 treated in step b), wherein the one or more DNA methylation markers comprise bases in a differentially methylated region (DMR) selected from the group consisting of BMP3, NDRG4, VAV3, SFMBT2, DIO3, HUNK, ELMO1, CD1D, CDKN2A, and OPLAH,

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

[0275] i) subjects who do not have Barrett's esophagus, to identify differences in the two sequences, and

[0276] ii) Subjects without Barrett's esophagus dysplasia to identify differences in the two sequences

[0277] e) When the difference in i) and ii) exists, the subject is identified as having Barrett's esophagus.

[0278] 27. The method of embodiment 26, wherein the determination of elevated methylation in one or more DNA methylation markers comprises the determination of altered methylation in regions selected from CpG islands and CpG island shores.

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

[0280] 29. A method as described in embodiment 26, wherein determining the methylation level of one or more DNA methylation markers in the DNA that has been processed in step b) includes determining the methylation score and / or 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. A method as described in embodiment 26, wherein the determining 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 following: 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 whole 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 the marker in a sample obtained from the subject; and

[0287] b) identifying the subject as

[0288] i) 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 Barrett's esophagus dysplasia, wherein the marker comprises bases in a differentially methylated region (DMR) selected from the group consisting of 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) identifying the subject as having Barrett's esophagus dysplasia when the methylation state of the marker is different from the methylation state of the marker determined in a subject with Barrett's esophagus or a subject without Barrett's esophagus dysplasia, wherein the marker comprises bases in a DMR selected from DMRs 3, 5, 30, 33, 43, 58, 77, 79-187;

[0290] iii) identifying the subject as having low-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 low-grade dysplasia of Barrett's esophagus, a subject without dysplasia of Barrett's esophagus, a subject with high-grade dysplasia of Barrett's esophagus, and a subject with esophageal adenocarcinoma, wherein the marker comprises bases in a DMR selected from the group consisting of 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 esophageal adenocarcinoma, wherein the marker comprises bases in a DMR selected from DMRs 77, 90, and 135; and

[0292] v) identifying the subject as having esophageal adenocarcinoma when the methylation state of the marker is different from the methylation state of the marker determined in a subject without esophageal adenocarcinoma, a subject without Barrett's esophagus dysplasia, 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 DMR 77, 90, and 135.

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

[0294] 36. The method according to embodiment 34,

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

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

[0297] When the methylation status of the marker is different from the methylation status of the marker determined in a subject who does not have Barrett's esophagus or a subject who does not have Barrett's esophagus dysplasia, the subject is identified as having Barrett's esophagus.

[0298] 37. The method according to 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 via a sponge capsule,

[0301] When the methylation status of the marker is different from the methylation status of the marker determined in a subject who does not have Barrett's esophagus or a subject who does not have Barrett's esophagus dysplasia, the subject is identified as having Barrett's esophagus.

[0302] 38. The method according to claim 34,

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

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

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

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

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

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

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

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

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

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

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

[0314] 48. The method of embodiment 34, wherein the marker is a 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 stool 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 determining comprises the use of methylation-specific oligonucleotides.

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

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

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

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

[0323] 57. The method of embodiment 34, wherein the determining comprises using 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 the group consisting of SEQ ID NOs: 1-50.

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

[0326] 60. The method of embodiment 34, wherein the chromosomal region having the annotations 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 Nos. 1-78.

[0328] 62. The method of embodiment 34, wherein the DMR is from Table 2 and is selected from DMR Nos. 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 Nos. 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 Nos. 77, 90, and 135.

[0331] 65. The method of embodiment 34, wherein the DMR is from Table 6 and is selected from DMR Nos. 136-187.

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

[0333] 67. The method of embodiment 34, wherein the DMR is from Table 8 and is 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 the sequence of a DMR selected from DMRs 1-229 from Tables 1, 2, 3, 5, 6, 7, and 8, and having a methylation state associated with a subject not having 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 state associated with a subject having Barrett's esophagus, Barrett's esophagus dysplasia, Barrett's esophagus low-grade dysplasia, Barrett's esophagus high-grade dysplasia, and esophageal adenocarcinoma.

[0342] 72. A kit comprising: a sample collector for obtaining a sample from a subject; reagents 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 the group consisting of 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 DMRs from Tables 1, 7, and 8;

[0349] b) comparing the methylation status of the marker in the sample from the subject with the methylation status of the marker in a normal control sample from a subject who does not have Barrett's esophagus or Barrett's esophagus dysplasia;

[0350] c) determining a confidence interval and / or p-value for the difference in methylation status between 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 Barrett's esophagus dysplasia 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 in a DMR selected from the group consisting of DMRs 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 in the sample from the subject with the methylation status of the marker in a normal control sample from a subject who does not have Barrett's esophagus dysplasia or Barrett's esophagus;

[0355] c) determining a confidence interval and / or p-value for the difference in methylation status between 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 low-grade dysplasia of Barrett's esophagus 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 in a DMR selected from DMR77, 90 and 135 from Table 5;

[0359] b) comparing the methylation status of the marker in the sample from the subject with the methylation status of the marker in normal control samples from subjects without low-grade dysplasia of Barrett's esophagus, subjects without dysplasia of Barrett's esophagus, subjects with high-grade dysplasia of Barrett's esophagus, and subjects with esophageal adenocarcinoma;

[0360] c) determining a confidence interval and / or p-value for the difference in methylation status between 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 whole esophageal swabbing or brushing or using a sponge capsule device.

[0364] 85. A method for screening for high-grade dysplasia of Barrett's esophagus 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 DMR77, 90 and 135 from Table 5;

[0366] b) comparing the methylation status of the marker in the sample from the subject with the methylation status of the marker in normal control samples from subjects without high-grade dysplasia of Barrett's esophagus, subjects without dysplasia of Barrett's esophagus, subjects with low-grade dysplasia of Barrett's esophagus, and subjects with esophageal adenocarcinoma;

[0367] c) determining a confidence interval and / or p-value for the difference in methylation status between 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 whole esophageal swabbing or brushing or using a sponge capsule device.

[0371] 89. A method for screening for esophageal adenocarcinoma 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 DMR77, 90 and 135 from Table 5;

[0373] b) comparing the methylation status of the marker in the sample from the subject with the methylation status of the marker in normal control samples from subjects without esophageal adenocarcinoma, subjects without Barrett's esophagus dysplasia, subjects with low-grade dysplasia of Barrett's esophagus, and subjects with high-grade dysplasia of Barrett's esophagus;

[0374] c) determining a confidence interval and / or p-value for the difference in methylation status between 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 whole 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 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) 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; and comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to the nucleotide sequence of a nucleic acid containing the DMR from:

[0379] a) subjects who do not have Barrett's esophagus, to identify differences in the two sequences, and

[0380] b) subjects without Barrett's esophagus dysplasia, to identify differences in the two sequences; and

[0381] When the difference in a) and b) exists, the subject is identified 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 dysplasia 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; and comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to the nucleotide sequence of a nucleic acid containing the DMR from:

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

[0385] b) subjects without Barrett's esophagus dysplasia, to identify differences in the two sequences; and

[0386] When the difference in a) and b) is present, the subject is identified as having Barrett's esophagus dysplasia.

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

[0388] 97. A method for screening for low-grade dysplasia of Barrett's esophagus in a sample obtained from a subject, the method comprising reacting a nucleic acid comprising a DMR (e.g., DMR Nos. 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; and comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to the nucleotide sequence of a nucleic acid containing the DMR from:

[0389] a) subjects without low-grade dysplasia of Barrett's esophagus, to identify differences in the two sequences,

[0390] b) subjects without Barrett's esophagus dysplasia, to identify differences in the two sequences,

[0391] c) subjects with high-grade dysplasia of Barrett's esophagus to identify differences in the two sequences, and

[0392] d) subjects with esophageal adenocarcinoma, to identify differences between the two sequences; and

[0393] When the differences in a), b), c), and d) are present, the subject is identified as having low-grade dysplasia of Barrett's esophagus.

[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 whole esophageal swabbing or brushing or using a sponge capsule device.

[0396] 100. A method for screening for high-grade dysplasia of Barrett's esophagus in a sample obtained from a subject, the method comprising reacting a nucleic acid comprising a DMR (e.g., DMR Nos. 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; and comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to the nucleotide sequence of a nucleic acid containing the DMR from:

[0397] a) subjects without high-grade dysplasia of Barrett's esophagus, to identify differences in the two sequences,

[0398] b) subjects without Barrett's esophagus dysplasia, to identify differences in the two sequences,

[0399] c) subjects with low-grade dysplasia of Barrett's esophagus to identify differences in the two sequences, and

[0400] d) subjects with esophageal adenocarcinoma, to identify differences between the two sequences; and

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

[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 whole 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 Nos. 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; and comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to the nucleotide sequence of a nucleic acid containing the DMR from:

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

[0406] b) subjects without Barrett's esophagus dysplasia, to identify differences in the two sequences,

[0407] c) subjects with low-grade dysplasia of Barrett's esophagus to identify differences in the two sequences, and

[0408] d) subjects with high-grade dysplasia 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 whole 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 with the methylation status of a control sample or reference sample recorded in a database; and an alarm component configured to determine a single value based on a combination of methylation states and alert a user of a methylation status associated with Barrett's esophagus.

[0413] 107. The system of embodiment 106, wherein the sample comprises a nucleic acid comprising a DMR.

[0414] 108. The system of embodiment 107, wherein the DMR is selected from the group consisting of 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 containing 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 Barrett's esophagus dysplasia.

[0420] 114. A system for screening for Barrett's esophagus dysplasia 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 with the methylation status of a control sample or reference sample recorded in a database; and an alarm component configured to determine a single value based on a combination of methylation states and alert a user of a methylation status associated with Barrett's esophagus dysplasia.

[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 nucleic acids.

[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 esophageal tissue.

[0426] 120. The system of embodiment 114, wherein the database comprises nucleic acid sequences containing DMRs.

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

[0428] 122. A system for screening for low-grade dysplasia of 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 with the methylation status of a control sample or reference sample recorded in a database; and an alarm component configured to determine a single value based on a combination of methylation states and alert a user of a methylation status associated with low-grade dysplasia of Barrett's esophagus.

[0429] 123. The system of embodiment 122, wherein the sample comprises a nucleic acid comprising a DMR.

[0430] 124. The system of embodiment 123, wherein the DMR is 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 whole esophageal swabbing or brushing or using a sponge capsule device.

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

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

[0437] 131. A system for screening for high-grade dysplasia of 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 with the methylation status of a control sample or reference sample recorded in a database; and an alarm component configured to determine a single value based on a combination of methylation states and alert a user of a methylation status associated with high-grade dysplasia of Barrett's esophagus.

[0438] 132. The system of embodiment 131, wherein the sample comprises a nucleic acid comprising a DMR.

[0439] 133. The system of embodiment 132, wherein the DMR is selected from DMR Nos. 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 whole esophageal swabbing or brushing or using a sponge capsule device.

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

[0445] 139. The system of embodiment 131, wherein the database comprises nucleic acid sequences from subjects with high-grade dysplasia of Barrett's esophagus, from subjects without Barrett's esophagus, from subjects without high-grade dysplasia of Barrett's esophagus, from subjects without low-grade dysplasia of Barrett's esophagus, 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 the methylation state of the sample; a software component configured to compare the methylation state of the sample with the methylation states of control samples or reference samples recorded in a database; and a software component configured to determine a single value based on a combination of methylation states and alert a user of a methylation state associated with esophageal adenocarcinoma.

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

[0448] 142. The system of embodiment 141, wherein the DMR is selected from DMR No. 77, 90, and 135.

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

[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 whole esophageal swabbing or brushing or using a sponge capsule device.

[0453] 147. The system of embodiment 140, wherein the database comprises nucleic acid sequences containing 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 high-grade dysplasia of Barrett's esophagus, from subjects not having low-grade dysplasia of Barrett's esophagus, 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 that does not have Barrett's esophagus, Barrett's esophagus dysplasia, Barrett's esophagus low-grade dysplasia, 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 related to 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 an agent 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 treated in step b), wherein the one or more DNA methylation markers comprise bases in differentially methylated regions (DMRs) 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 with a reference methylation level of the one or more DNA methylation markers of:

[0465] i) subjects who do not have Barrett's esophagus, to identify differences in the two sequences, and

[0466] ii) Subjects without Barrett's esophagus dysplasia to identify differences in the two sequences

[0467] e) When the difference in i) and ii) exists, the subject is identified as having Barrett's esophagus.

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

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

[0470] 157. A method as described in embodiment 154, wherein determining the methylation level of one or more DNA methylation markers in the DNA that has been processed in step b) includes determining the methylation score and / or methylation frequency of the one or more DNA methylation markers.

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

[0472] 159. A method as described in embodiment 154, wherein the determining 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 following: 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 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 an agent 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 processed in step b), wherein the one or more DNA methylation markers comprise bases in differentially methylated regions (DMRs) 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 with a reference methylation level of the one or more DNA methylation markers of:

[0479] i) subjects with Barrett's esophagus, to identify differences in the two sequences, and

[0480] ii) subjects without Barrett's esophagus dysplasia, to identify differences in the two sequences; and

[0481] e) when the difference in i) and ii) is present, identifying the subject as having Barrett's esophagus dysplasia.

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

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

[0484] 164. A method as described in embodiment 161, wherein determining the methylation level of one or more DNA methylation markers in the DNA that has been processed in step b) includes determining the methylation score and / or methylation frequency of the one or more DNA methylation markers.

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

[0486] 166. A method as described in embodiment 161, wherein the determining 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 following: 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 low-grade dysplasia of Barrett's esophagus 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 methylation level of one or more DNA methylation markers in the DNA that has been processed 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,

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

[0493] i) subjects without low-grade dysplasia of Barrett's esophagus, to identify differences in the two sequences,

[0494] ii) subjects without Barrett's esophagus dysplasia, to identify differences in the two sequences,

[0495] iii) subjects with high-grade dysplasia of Barrett's esophagus to identify differences in the two sequences, and

[0496] iv) subjects with esophageal adenocarcinoma, to identify differences between the two sequences; and

[0497] e) When the differences in i), ii), iii, and iv) are present, the subject is identified as having low-grade dysplasia of Barrett's esophagus.

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

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

[0500] 171. A method as described in embodiment 168, wherein determining the methylation level of one or more DNA methylation markers in the DNA that has been processed in step b) includes determining the methylation score and / or methylation frequency of the one or more DNA methylation markers.

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

[0502] 173. A method as described in embodiment 168, wherein the determining 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 following: 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 whole esophageal swabbing or brushing or using a sponge capsule device.

[0505] 176. A method for detecting 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 a 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 processed 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,

[0509] d) comparing the determined methylation level of the one or more DNA methylation markers with a reference methylation level of the one or more DNA methylation markers of:

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

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

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

[0513] iv) subjects with esophageal adenocarcinoma, to identify differences between the two sequences; and

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

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

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

[0517] 179. A method as described in embodiment 176, wherein determining the methylation level of one or more DNA methylation markers in the DNA that has been processed in step b) includes determining the methylation score and / or 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. A method as described in embodiment 176, wherein the determining 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 following: 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 whole 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 processed 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,

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

[0527] i) subjects who do not have esophageal adenocarcinoma, to identify differences in the two sequences,

[0528] ii) subjects without Barrett's esophagus dysplasia, to identify differences in the two sequences,

[0529] iii) subjects with low-grade dysplasia of Barrett's esophagus to identify differences in the two sequences, and

[0530] iv. subjects with high-grade dysplasia of Barrett's esophagus to identify differences in the two sequences; and

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

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

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

[0534] 187. A method as described in embodiment 184, wherein determining the methylation level of one or more DNA methylation markers in the DNA that has been processed in step b) includes determining the methylation score and / or methylation frequency of the one or more DNA methylation markers.

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

[0536] 189. A method as described in embodiment 184, wherein the determining 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 following: 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 whole esophageal swabbing or brushing or using a sponge capsule device.

[0539] Example

[0540] Example 1.

[0541] Molecular markers may aid in the detection of Barrett's esophagus (BE) and monitoring of BE-related dysplasia (BED) by endoscopic or nonendoscopic methods. Experiments were conducted to (1) identify and validate novel methylated DNA markers for BE dysplasia and (2) test the feasibility of candidate markers for detecting BE dysplasia from whole esophageal brushings.

[0542] Using global methylome bisulfite sequencing of DNA from BE tissues without dysplasia, with low-grade dysplasia (BE-LGD), with high-grade dysplasia (BE-HGD), or adenocarcinoma (EAC) (18 samples per group), candidate markers were identified to separate BE from normal tissue, and to separate BED from BE without dysplasia.

[0543] Tables 1 and 7 provide the DMR information of such markers identified to separate BE and normal tissues, including chromosome number, gene annotation, and DMR start / end position (see Example II for Materials / Methods for generating Tables 1 and 7).

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

[0545] A high-capacity cytology brush (Hobbs Medical, Stafford Springs, CT) was used to perform whole esophageal brushing on informed consent patients with BE who were planning endoscopic BE monitoring or endoscopic evaluation of BE-related cancers. Sampling was performed from the cardia circumference over the entire esophagus length (BE+squamous mucosa) to simulate swallowing using a sponge-on-a-string device. 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 (a marker of total human DNA). 12 abnormally methylated genes were identified that best distinguished BED from BE without dysplasia (e.g., area under the ROC curve 0.86-0.97) (see Table 3). These 12 markers are DIO3, MAX20.218, CD1D, T-SPYL5, ZNF568, ST8SIA1, ELMO1, ELOVL2, BMP3, NDRG4, HUNK, and CDKN2A. Table 3 DMRs provides information on the identification of such markers to separate BED from BE without dysplasia in esophageal samples obtained by whole esophageal brushing, including chromosome number, gene annotation, and DMR start / end position. Table 4 provides forward and reverse primer information for the DMRs provided in Table 3. Thirty-nine subjects were studied, with a median age of 69 years (range, 28-94 years), 74% male, and a median BE length of 4 (1-14) cm; 18 had no dysplasia, and 21 had dysplasia (9 LGD, 7 HGD, and 5 EAC (4 asymptomatic early stage). The panel of three markers (DIO3, MAX20.218, NDRG4) (see Table 5) detected 78% of LGD, 71% of HGD, 100% of EAC, and 81% of all dysplasia at 95% specificity (see Table 5). Figure 1 Table 5 provides the DMR information of such markers identified to distinguish LGD, HGD and EAC, including chromosome number, gene annotation and DMR start / end position.

[0546] Table 1. Information about DMRs that differentiate BE and normal tissues

[0547]

[0548]

[0549]

[0550] Table 2. Information on DMRs that differentiate BE from BED

[0551]

[0552]

[0553]

[0554] Table 3 - Information on DMRs that differentiate BE from BED

[0555]

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

[0557]

[0558]

[0559] Table 5 - Information on DMRs that differentiate LGD, HGD, and EAC, and BE from BED

[0560]

[0561] Table 6 - Information on DMRs that differentiate BE from BED

[0562]

[0563]

[0564]

[0565] Table 7 - Information on DMRs that differentiate BE from normal tissues

[0566]

[0567] Example II.

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

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

[0570] Library preparation: Genomic DNA (300 ng) was fragmented by digestion with 10 units of MspI (a methylation-specific restriction enzyme that recognizes 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' exosome), and ligated overnight with methylated TruSeq adapters (Illumina, San Diego, CA) containing barcode sequences (each fragment was ligated 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). The buffer cutoff was 0.7X-1.1X the sample volume of beads / buffer. The final elution volume was 22 uL (EB buffer - Qiagen, Germantown, MD); qPCR was used to measure ligation efficiency and fragment quality on a small sample aliquot. The samples were then bisulfite converted (twice) using a modified EpiTect protocol (Qiagen). qPCR and conventional PCR (PfuTurbo Cx hotstart–Agilent, Santa Clara, CA) performed on aliquots of the converted samples, followed by evaluation on a Bioanalyzer 2100 (Agilent), determined the optimal number of PCR cycles before final library amplification. The following conditions were used for the final PCR: 1.) Each 50 μL reaction contained 5 μL of 10X buffer, 1.25 μL of 10 mM of each deoxyribonucleoside triphosphate (dNTP), 5 μL of primer mix (~5 μM), 15 μL of template (sample), 1 μL of PfuTurbo Cx hotstart, and 22.75 of water; the temperature and time were 95°C for 5 minutes; 98°C for 30 seconds; and 16 cycles of 98°C for 10 seconds, 65°C for 30 seconds, 72°C for 30 seconds, 72°C for 5 minutes, and a hold at 4°C. Samples were combined (equimolar) into 4-plex libraries based on a randomization scheme and tested as follows: final size verification using a bioanalyzer and qPCR using phiX standards and adapter-specific primers.

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

[0572] MSP primer design: Primers for the six best markers from the sequencing results were designed and sequenced (IDT, Coralville, IA) to target specific bisulfite-modified methylated sequences (Table 7). These designs were completed using Methprimer software (University of California, San Francisco, CA) or MSP Primer (Johns Hopkins University, Baltimore, MD). The assay was tested and optimized by qPCR using SYBR Green on dilutions of ubiquitously methylated and unmethylated genomic DNA controls.

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

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

[0575] For the RRBS discovery phase, the primary comparison of interest was methylation differences between Barrett's cases, esophageal controls, and leukocyte controls for each mapped CpG. CpG islands were defined biochemically by observed expected CpG ratios > 0.6. (30) However, for this model, tiling units for CpG analysis, “differentially methylated regions (DMRs),” were generated based on the distance between CpG site locations on each chromosome. Islands with only a single CpG were excluded. Single CpG sites were considered for differential analysis only if the total coverage depth per disease group was ≥ 200 reads (average 10 reads / subject) and the variance of % methylation was > 0 (excluding non-informative 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 read counts. To account for variations in read depth across individual subjects, an overdispersion logistic regression model was used, with the dispersion parameter estimated using the Pearson chi-square statistic of the residuals from the fitted model. DMRs ranked by their significance level were further considered if the % methylation in benign esophageal and leukocyte controls and in Barrett's cases was ≤1% but ≥10%. 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 six markers (Table 7), the primary outcome was the area under the receiver operating characteristic curve (AUC) for each marker, as calculated from a logistic regression model of the % methylated copy number for each sample with BE compared to normal esophagus and normal leukocytes. For each marker, the AUC was again > 0.90, and the quantitative difference in the mean number of candidate genome copies per sample between cases and controls was at least 50-fold.

[0577] Example III.

[0578] This example describes the materials and methods used to produce Table 2 and Table 6.

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

[0580] Library preparation: Genomic DNA (300 ng) was fragmented by digestion with 10 units of MspI (a methylation-specific restriction enzyme that recognizes 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' exosome), and ligated overnight with methylated TruSeq adapters (Illumina, San Diego, CA) containing barcode sequences (each fragment was ligated 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). The buffer cutoff was 0.7X-1.1X the sample volume of beads / buffer. The final elution volume was 22 uL (EB buffer - Qiagen, Germantown, MD); qPCR was used to measure ligation efficiency and fragment quality on a small sample aliquot. The samples were then bisulfite converted (twice) using a modified EpiTect protocol (Qiagen). qPCR and conventional PCR (PfuTurbo Cx hotstart–Agilent, Santa Clara, CA) performed on aliquots of the converted samples, followed by evaluation on a Bioanalyzer 2100 (Agilent), determined the optimal number of PCR cycles before final library amplification. The following conditions were used for the final PCR: 1.) Each 50 μL reaction contained 5 μL of 10X buffer, 1.25 μL of 10 mM of each deoxyribonucleoside triphosphate (dNTP), 5 μL of primer mix (~5 μM), 15 μL of template (sample), 1 μL of PfuTurbo Cx hotstart, and 22.75 of water; the temperature and time were 95°C for 5 minutes; 98°C for 30 seconds; and 16 cycles of 98°C for 10 seconds, 65°C for 30 seconds, 72°C for 30 seconds, 72°C for 5 minutes, and a hold at 4°C. Samples were combined (equimolar) into 4-plex libraries based on a randomization scheme and tested as follows: final size verification using a bioanalyzer and qPCR using phiX standards and adapter-specific primers.

[0581] Sequencing and Bioinformatics: Samples were loaded onto the flow cell according to random lane assignment, with additional lanes reserved for internal assay controls. Sequencing was performed at the Mayo Clinic Medical Genome Facility on an Illumina HiSeq 2000 using the Next Generation Sequencing Core. Reads were unidirectional for 101 cycles. Each flow cell lane generated 100 million to 120 million reads, sufficient to align sequences to a median coverage of 30–50× sequencing depth (number of reads per CpG). Standard Illumina pipeline software accessed bases and generated sequencing reads in fastq format. As previously described, (28) SAAP-RRBS (streamlined analysis and annotation pipeline for 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 sequenced (IDT, Coralville IA) to target specific bisulfite-modified methylated sequences (Table 7). These designs were completed using Methprimer software (University of California, San Francisco CA) or MSP Primer (Johns Hopkins University, Baltimore, MD). The assay was tested and optimized by qPCR using SYBR Green on dilutions of ubiquitously 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-grade dysplasia, 36 with low-grade dysplasia, and 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 differential % methylation between cases and controls, and based on the area under the receiver operating characteristic curve (AUC) and target to background ratio.

[0585] For the RRBS discovery phase, the primary comparison of interest was the methylation differences between Barrett's cases with high and low dysplasia, Barrett's controls without dysplasia, and leukocyte controls for each mapped CpG. CpG islands were defined biochemically by an observed expected CpG ratio > 0.6. (30) However, for this model, tiling units for CpG analysis, “differentially methylated regions (DMRs),” were generated based on the distance between CpG site locations on each chromosome. Islands with only a single CpG were excluded. Single CpG sites were considered for differential analysis only if the total coverage depth per disease group was ≥ 200 reads (average 10 reads / subject) and the variance of % methylation was > 0 (excluding non-informative 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 read counts. To account for variations in read depth across individual subjects, an overdispersion logistic regression model was used, with the Pearson chi-square statistic of the residuals from the fitted model estimating the discrete parameters. If the % methylation in benign esophageal and leukocyte controls was <1% but >10% in Barrett's cases, DMRs ranked by their significance level were further considered. This resulted in 57 markers (Table 2). All had AUCs ranging from 0.60 to 0.87, and fold changes ranging from 2 to 10. A second sorting of the data was performed, relaxing island-restricted measurements, and focusing on grouping of highly discriminative single CpGs. The inter-batch effect on leukocyte controls was also removed, which increased overall coverage. This resulted in 52 additional markers with increased fold changes (7-52) and similar AUCs (Table 6).

[0586] For the 66 DMRqMSP validation studies, the primary outcome was the area under the receiver operating characteristic curve (AUC) for each marker, as calculated from a logistic regression model of the % methylated copy number per sample with BED compared to BE and normal leukocytes. Twelve markers demonstrated excellent performance (Table 3). AUCs ranged from 0.86 to 0.97, with fold changes ranging from 2 to 24. Ten of the 12 markers were included in the esophageal brushing feasibility study along with BMP3 and NDRG4.

[0587] Example IV.

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

[0589] Informed consenting BE patients scheduled for endoscopic BE surveillance or endoscopic evaluation of BE-related cancers were subjected to whole esophageal brushing using a high-capacity cytology brush (Hobbs Medical, Stafford Springs, CT), sampling circumferentially from the cardia through the entire esophagus length (BE + squamous mucosa). The cytology brush was removed from the handle and placed in a vial of stabilization / cell lysis buffer 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 on 10 of the 12 validated DMRs from the BED vs BE study (Table 3) using 20 ng of converted DNA. Primer sequences are highlighted in Table 4. In addition, BMP3 and NDRG4 Cologuard QuARTs assays were performed. The AUCs were compared and the significance of the differences was measured using the DeLong, DeLong, and Clarke-Pearson methods. Bonferroni correction was used to avoid bias from multiple comparisons. The three markers (combined) 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 a methylated DNA marker for the detection of Barrett's esophagus.

[0592] Phase 1 Methods and Results:

[0593] Pathologist-verified FFPE tissues were provided by the Mayo Clinic Tissue Registry. The clinical group consisted of patients with the following: 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 the Qiagen Mini kit and quantified by absorbance and picogreen analysis. Bisulfite conversion was performed using the Zymo method. Methylation markers consisted of the best candidates from three RRBS subsets: 1) 45 BED vs. BE DMRs (differentially methylated regions), 2) 5 BE vs. normal esophageal DMRs, and 3) 33 previously validated esophageal cancer markers. (Note: All of these DMRs were previously filtered for normal leukocytes for background methylation <1%). Methylation-specific PCR (MSP) primers were designed for each of these genomic regions and performance tested on three sets of methylation controls. Table 8 provides the DMR information for such markers identified to separate BE and normal tissues, including chromosome number, gene annotation, and DMR start / end position. 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 four included two reference genes, β-actin and ZDHHC1, in their triple assay format.

[0594] Results were normalized to β-actin and ZDHHC1, and logistic analysis was performed in JMP. The area under the receiver operating characteristic (ROC) curve (AUC) was calculated, as well as the fold change and p-value. The performance cutoffs for stage 2 were AUC ≥ 0.95, fold change ≥ 25, and p-value ≤ 0.1. Thirteen markers met these criteria: CDKN2A, SFMBT2, VAV3, DIO3, ELMO1, FEM1B, HUNK, ADCY1, CD1D, ST3GAL6, LRRC4, NDRG4, and BMP3 (Table 9 provides the identities and primer sequences for these assays, including OPLAH).

[0595] Table 8. Information about DMRs that differentiate BE from normal tissues

[0596]

[0597]

[0598]

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

[0600]

[0601] Phase 2 Methods and Results

[0602] 49 cases with BE and 36 controls without BE were recruited before endoscopy. The median age was 69 years (range 63-73 years) and 59 years (45-67 years), with 92% and 42% male, respectively. BE cases had >1 cm (median number = 2 cm; IQR 4-8) of circumferential columnar mucosa with confirmed intestinal metaplasia; controls did not have BE on endoscopy. Samples were obtained using a high-capacity endoscopic cytology brush (Hobbs Medical, Stafford Springs CT); a sponge sampling device that swabbed the cardia, BE (cases), and the entire esophagus to simulate swallowing. The brush was placed in a 2 ml vial containing lysis buffer and quickly frozen at -80°C. The samples were thawed and processed as batches in a blinded manner. After the vial was vigorously vortexed to remove all cellular material from the brush, DNA was purified using the Gentra Puregene kit (Qiagen). This method allows for the simultaneous harvesting of free DNA and cellular DNA. The samples were then treated with sodium bisulfite and recovered using the EZ DNA Methylation Kit (ZymoResearch).

[0603] Methylation of 13 target genes was determined by QMSP and QuARTs on Roche 480 LightCyclers as before. 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; the distribution of methylation levels from BE cases and controls was significantly different ( Figure 2 ). Methylation levels were correlated with BE length and the presence of dysplasia, p < 0.05. Figure 3 A hit matrix of the best methylated DNA markers from Phase 2 highlighting complementarity (endoscopic brush study) is shown.

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

[0605] Phase 3 Methods and Results:

[0606] In 10 cases with BE and 12 controls without obvious BE, a capsule sponge device (EsophaCap, Capnostics) was swallowed and removed, followed by endoscopy within 24 hours. In the 10 cases and 12 controls, the median age was 65 years (59-69 years) and 40 years (34-61 years), respectively, with males accounting for 70% and 45%, 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 additional aliquots of PreservCyt for a total of 40 ml. The cells were pelleted and lysed in 1 mL of buffer (Puregene Buccal Cell Kit) and extracted according to the manufacturer's instructions. A second extraction method (Maxwell-Promega) was also tested. After bisulfite conversion (Zymo Research), the samples were assayed by QPCR as before. The profile of the best markers from the sponge was highly discriminatory for BE. At 100% specificity, a panel of markers detected all 9 BE cases that met the inclusion criteria (100% sensitivity) (1 did not meet the inclusion criteria).

[0607] Figure 4 Shown are the levels of methylated DNA markers (PCR copies / 30 ng DNA) in BE cases and normal (N1) controls from Phase 3 (capsule sponge study).

[0608] The entire contents of all publications and patents mentioned in the above description are incorporated herein by reference for all purposes. Various modifications and variations of the described compositions, methods and uses of the present technology will be apparent to those skilled in the art without departing from the scope and spirit of the described technology. Although the present technology has been described in conjunction with specific exemplary embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, various modifications of the described modes for implementing the present invention that are apparent to those skilled in the art of pharmacology, biochemistry, medical science or related fields are intended to be within the scope of the appended claims.

Claims

1. Use of a methylation-specific reagent and a primer set for specifically detecting a differentially methylated region in ZNF682 in the preparation of a kit for identifying esophageal disease in a sample from a subject by determining the methylation level of the differentially methylated region in ZNF682; wherein the differentially methylated region comprises at least one CpG site from chromosome region 20149796-20149923 of chromosome 19, and the chromosome region 20149796-20149923 can be amplified using a primer pair comprising SEQ ID NOs: 31 and 32 after treatment with a methylation-specific reagent; and wherein the esophageal disease is Barrett's esophagus or Barrett's esophagus dysplasia.

2. The use according to claim 1, wherein the kit further comprises a primer set for specifically detecting differentially methylated regions in NDRG4.

3. The use according to claim 1, wherein the kit further comprises a primer set for specifically detecting differentially methylated regions in VAV3.

4. The use according to claim 1, wherein the kit further comprises a primer set for specifically detecting differentially methylated regions in NDRG4 and a primer set for specifically detecting differentially methylated regions in VAV3.

5. The use according to claim 1, wherein the kit further comprises a primer set for specifically detecting differentially methylated regions in at least one additional gene selected from CD1D, DIO3, HUNK, CDKN2NA, BMP3, ELMO1, SFMBT2 and OPLAH.

6. The use according to claim 1, wherein the esophageal disease is Barrett's esophagus-associated low-grade dysplasia or Barrett's esophagus-associated dysplasia-high-grade dysplasia.

7. The use according to claim 1, wherein the sample is a tissue sample.

8. The use of claim 7, wherein the tissue sample comprises esophageal tissue obtained by whole esophageal swabbing or brushing.

9. The use according to claim 1, wherein the methylation level of the differentially methylated region in ZNF682 comprises an increased methylation level relative to a normal methylation level.

10. The use of claim 1, wherein the methylation level of the differentially methylated region in ZNF682 comprises a different pattern of methylation relative to a normal methylation pattern.

11. The use according to claim 1, wherein determining the methylation level of the differentially methylated region in ZNF682 comprises using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation and / or target capture. 12 . The method according to claim 1 , wherein determining the methylation level of the differentially methylated region in ZNF682 comprises determining a methylation score and / or a methylation frequency. 13 . The use according to claim 1 , wherein determining the methylation level of the differentially methylated region in ZNF682 comprises treating genomic DNA in the sample with a methylation-specific reagent. 14 . The use according to claim 1 , wherein the primer set for specifically detecting the differentially methylated region in ZNF682 comprises a primer pair having nucleotide sequences as shown in SEQ ID NOs: 31 and 32. 15 . The use according to claim 2 , wherein the primer set for specifically detecting the differentially methylated region in NDRG4 comprises a primer pair having nucleotide sequences as shown in SEQ ID NOs: 15 and 16. 16 . The use according to claim 3 , wherein the primer set for specifically detecting the differentially methylated region in VAV3 comprises a primer pair having nucleotide sequences as shown in SEQ ID NOs: 39 and 40.

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