Methods to differentiate barrett's esophagus disease stages

Abstract

A method for assessing Barrett's esophagus disease stage of a subject includes obtaining a sample from the subject and converting the sample to a modified sample. The method further includes analyzing the modified sample to identify a presence of at least one biomarker and determining the disease stage of the subject. The method further comprises determining the treatment regimen to treat the subject.

Description

Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 METHODS TO DIFFERENTIATE BARRETT'S ESOPHAGUS DISEASE STAGES CROSS-REFERENCED TO RELATED PATENT APPLICATIONS

[0001] This application claims the benefit of U.S. Patent Provisional Application No. 63 / 604,097, filed November 29, 2023, and U.S. Patent Provisional Application No.63 / 647,940, filed May 15, 2024, which are hereby incorporated by reference it their entireties. TECHNICAL FIELD

[0002] Aspects of the disclosure relate generally to genetic classifiers and methods using same to differentiate disease stages. Compositions and methods to differentiate late-stage Barrett’s Esophagus (BE) and Esophageal Adenocarcinoma (EAC) from early-stage disease are provided. In some embodiments, methods to treat non-dysplastic BE (NDBE) to low-grade dysplasia (LGD), to neoplastic high-grade dysplasia (HGD), and Esophageal Adenocarcinoma (EAC) are provided. BACKGROUND

[0003] Esophageal Adenocarcinoma (EAC) is the second deadliest cancer in the United States, with a grim five-year survival rate of less than 20%. Barrett's Esophagus (BE) is the precursor to EAC, with well-defined stages of disease progression. BE advances from Benign metaplasia, i.e., non-dysplastic BE (NDBE) to low-grade dysplasia (LGD), to neoplastic high- grade dysplasia (HGD), and finally to cancer. Although most NDBE cases do not become cancer, this predictable progression pattern provides an opportunity for surveillance and intervention at the dysplastic stage by highly effective endoscopic eradication modalities to prevent further progression to EAC. SUMMARY

[0004] Aspects of the disclosure relate to a method for identifying a Barrett’s esophagus (BE) disease stage, the method comprising: extracting nucleic acid molecules from an esophageal sample from a subject; applying targeted nucleic acid sequence enrichment and sequencing the nucleic acid molecules; quantifying an amount of copy number alterations (CNA) of fifty or more genes listed in Table 1, wherein the amount of CNA is defined as the number of copy number gains and losses within the nucleic acid, wherein if the amount is above a predetermined value, the disease stage is late stage of BE; and identifying whether the disease stage is early stage or late stage of BE. In some embodiments, the method comprises determining a CNA profile of the fifty of more genes listed in Table 1. In some embodiments,Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 the fifty or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the method further comprises quantifying presence of gene mutations of fifty or more genes listed in Table 1. In some embodiments, the method further comprises quantifying presence of TP53 gene mutations. In some embodiments, a significant increase in the CNA relative to the predetermined value is an indication that the subject has neoplastic high-grade dysplasia (HGD) or has esophageal adenocarcinoma (EAC). In some embodiments, a mean CNA value over the predetermined value is an indication that the subject has neoplastic high- grade dysplasia (HGD) or has esophageal adenocarcinoma (EAC). In some embodiments, the predetermined value is greater than 20. In some embodiments, the predetermined value is from 20 to 100. In some embodiments, the method further comprises treating the subject. In some embodiments, the treating comprises removing at least part of the esophagus of the subject.

[0005] Aspects of the disclosure relate to method for monitoring Barrett’ s esophagus (BE) in a subject, the method comprising quantifying in a nucleic acid sample of the subject an amount of copy number alterations (CNA) of fifty or more genes listed in Table 1, wherein the amount of CNA is defined as the number of copy number gains and losses within the nucleic acid sample, wherein the subject has non-dysplastic BE (NDBE) to low-grade dysplasia (LGD) if mean CNA is lower than a predetermined value. In some embodiments, the predetermined value is about 20. In some embodiments, the method further comprises monitoring BE in the subject. In some embodiments, the monitoring is every 3 to 5 years. In some embodiments, the monitoring comprises repeating the step of quantifying. In some embodiments, the monitoring comprises performing endoscopic surveillance.

[0006] Aspects of the disclosure relate to a method of characterizing DNA is a biological sample of a subject, the method comprising extracting DNA from the biological sample, wherein the biological sample comprises esophageal cells; fragmenting the DNA; constructing DNA fragment library; subjecting DNA fragments to hybridization capture; sequencing DNA fragments; aligning DNA sequences to reference human genome; determining mean CNA of fifty of more genes listed in Table 1; and using the mean CNA to determine if the DNA is derived from non-dysplastic BE (NDBE), low-grade dysplasia (LGD), neoplastic high-grade dysplasia (HGD), or Esophageal Adenocarcinoma (EAC) biological sample. In some embodiments, the mean CNA over a predetermined value is an indication that the subject has neoplastic high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC). In some embodiments, the predetermined value is greater than 20. In some embodiments, the predetermined value is from 20 to 100.Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24

[0007] Aspects of the disclosure relate to a method of treating Barrett’s esophagus (BE) in a subject in need thereof, comprising: obtaining a biological sample from the subject; extracting nucleic acid from the biological sample; analyzing the nucleic acid to quantify an amount of copy number alterations (CNA) of fifty or more genes listed in Table 1, wherein the amount of copy number alterations is defined as the number of copy number gains and losses; determining whether the subject has early stage or late stage BE, wherein mean CNA over a predetermined value is an indication that the subject has late stage BE; and monitoring or treating the subject according to the BE disease stage. In some embodiments, the predetermined value is greater than 20. In some embodiments, the predetermined value is from 20 to 100. In some embodiments, if the subject has non-dysplastic BE (NDBE) or low-grade dysplasia (LGD), the subject is monitored. method is repeated every 3 to 5 years. In some embodiments, if the subject has non-dysplastic BE (NDBE) or low-grade dysplasia (LGD), the method is repeated every 3 to 5 years. In some embodiments, if the subject has neoplastic high-grade dysplasia (HGD), proton pump inhibitors, antacids or H2 blockers, photodynamic therapy and endoscopic mucosal therapy. In some embodiments, if the subject has Esophageal Adenocarcinoma (EAC), the subjects is treated by endoscopic mucosal resection or removing at least part of the esophagus.

[0008] Aspects of the disclosure relate to a genetic panel for diagnosing a subject with non- dysplastic BE (NDBE), low-grade dysplasia (LGD), neoplastic high-grade dysplasia (HGD), or Esophageal Adenocarcinoma (EAC), wherein the genetic panel comprises 50 or more genes of Table 1. In some embodiments, the genetic panel comprises from 50 to 311 genes of Table 1. In some embodiments, the 50 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel is used to identify Barrett’s esophagus (BE) disease stage in a subject in need thereof. In some embodiments, the genetic panel is used to stratify subjects with BE.

[0009] In some embodiments, a method for assessing a disease stage of a subject includes obtaining a sample from the subject, converting the sample to a modified sample, and analyzing the modified sample to identify a presence of at least one biomarker. The method may further include determining the disease stage of the subject. In some embodiments, the at least one biomarker comprises at least one of copy number alternation (CNA), differential methylation, single nucleotide polymorphisms / insertions-deletions (SNP / Indel), microsatellite instability (MSI), differential RNA expression, and changes in protein expression. In some embodiments, converting the sample includes at least one of chemically, mechanically, or enzymatically converting the sample. In further embodiments, the at least one biomarker is differentialAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 methylation, and converting the sample includes converting the sample to differentiate methylated DNA bases from unmethylated DNA bases. In further embodiments, the at least one biomarker is CNA, and converting the sample includes at least one of reducing a size of DNA fragments within the sample and applying targeted enrichment of the sample. In further embodiments, the at least one biomarker is SNP / Indel, and converting the sample includes at least one of reducing a size of DNA fragments within the sample, applying targeted enrichment, and amplification of the sample. In further embodiments, the at least one biomarker is MSI, and converting the sample includes at least one of reducing a size of DNA fragments within the sample, applying targeted enrichment, and amplification of the sample. In some embodiments, the at least one biomarker is RNA expression or protein expression, and converting the sample includes at least one of reducing a size of DNA fragments within the sample, applying targeted enrichment, and amplification of the sample. In further embodiments, the disease stage qualifies the disease stage of Barrett’s Esophagus or esophageal cancer.

[0010] In another embodiment, a method for identifying a disease state of a sample from the subject includes obtaining the sample from the subject and analyzing the sample to determine a presence amount of at least one biomarker within the sample. The method may further include determining the disease state of the sample through comparing a presence of an amount of the at least one biomarker with at least one predetermined value. In some embodiments, the at least one predetermined value is at least one value between twenty and ninety events, and if the presence amount is above the at least one predetermined value, the disease state is late stage. In further embodiments, the at least one predetermined value is forty-eight events, and if the presence amount is above forty-eight events, the disease is late stage. In some embodiments, the at least one predetermined value is at least one value between twenty and ninety events, and when the presence amount is less than or equal to the at least one predetermined value, the disease state is early stage. In further embodiments, the at least one predetermined value is forty-eight events, and if the presence amount is below forty-eight events, the disease is early stage.

[0011] In another embodiment, a method for identifying a progression level of a disease of a sample includes obtaining the sample from a subject, converting the sample into a modified sample, measuring an amount of a target biomarker within the modified sample, the target biomarker comprising at least one of a copy number alternation (CNA) value, a single nucleotide polymorphisms / insertions-deletions (SNP / Indel) value, a microsatellite instability (MSI) value, a differential methylation value, a differential RNA expression value, and changesAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 in protein expression, and comparing a value of the target biomarker with a predetermined value wherein if the biomarker amount is below or equal to the predetermined value, a disease state of the sample is identified as early stage and if the biomarker is above the predetermined value, the disease state of the sample is identified as late stage. In some embodiments, converting the sample into a modified sample includes treating the sample such that unmethylated Cytosine bases are converted to Uracil bases. In further embodiments, the predetermined value is between twenty and ninety events.

[0012] In another embodiment, a method for identifying a disease state of a sample from a subject includes obtaining the sample from the subject, analyzing the sample from the subject, analyzing the sample to quantify an amount of copy number alterations (CNA) within the sample, wherein the amount of copy number alterations is defined as the number of copy number gains and losses within the sample, and identifying whether the disease state of the sample is early stage or late stage. In some embodiments, the method includes analyzing the sample to quantify a presence of gene mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 is a graph displaying mean copy number alteration values for early-stage disease samples and late-stage disease samples, in accordance with Example 1.

[0014] FIG.2 is a graph illustrating the difference in mean number of high impact mutations between early and late stage of the disease for a variety of genes, in accordance with Example 1.

[0015] FIG. 3 is a graph displaying the AUC-ROC curve for copy number alterations, in accordance with Example 1.

[0016] FIG.4 is a illustrates the AUC-ROC curve for copy number alterations combined with TP53 COSMIC mutations, in accordance with Example 1.

[0017] FIG.5 is a table displaying performance metrics of the disease stratification classifier for copy number alterations and the combination of copy number alterations and TP53 COSMC mutations, in accordance with Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] Various exemplary embodiments will be described more fully hereinafter with reference to the accompanying drawings, in which some example embodiments are shown. The present inventive concept may, however, be embodied in many different forms and should notAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 be construed as limited to the example embodiments set forth herein. Rather, these example embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present inventive concept to those skilled in the art.

[0019] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this inventive concept belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0020] The term “esophagus” is intended to encompass the upper portion of the digestive system spanning from the back of the oral cavity, passing downwards through the rear part of the mediastinum, through the diaphragm and into the stomach. Chronic Gastroesophageal reflux disease (GERD) represents reflux condition in which stomach’s acidic content move up to esophagus, which increases the risk for developing esophageal cancer. The term “esophageal cancer” is used herein to refer to any cancerous neoplasia of the esophagus. “Barrett's esophagus” as used herein refers to an abnormal change (metaplasia) in the cells of the lower portion of the esophagus. Barrett's is characterized the finding of intestinal metaplasia in the esophagus. “Dysplasia” means presence of cells of abnormal type in a tissue which may or may not develop cancer. “Non dysplastic Barret Esophagus” (NDBE) refers to Barrett esophagus where no pre-cancerous stages are found. “High Grade Dysplasia” (HGD) refers to more advanced stages of Esophagus dysplasia for developing cancerous stage than “LGD” or Low- Grade Dysplasia. The term “esophageal cancer” or “EAC” is used herein to refer to any cancerous neoplasia of the esophagus.

[0021] The term “sample collection” or “brushing” of the esophagus, as referred to herein, may be obtained using any of the means known in the art. In some embodiments, a brushing is obtained by contacting the esophagus with a brush, a cytology brush, a sponge, a balloon, or with any other device or substance that contacts the esophagus and obtains an esophageal sample.

[0022] The term “biomarker” is meant to include, but is not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, natural product extract libraries, and any other molecules (including, but not limited to, DNA, RNA, Protein, miRNA). The presence of a biomarker beyond a limit is indicative of onset of a disease or advanced disease stage.Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24

[0023] The term “modified sample” herein refers to genetic material that has been treated or reacted with a chemical compound, enzyme, or by any form of mechanical shearing, that converts sample material to allow analysis of any one of the biomarkers. For example, one such compound is sodium bisulfite, which converts unmethylated C to U. If DNA that contains conversion-sensitive cytosine is treated with sodium bisulfite, the compound-converted DNA will contain U in place of C. If the DNA which is treated with sodium bisulfite contains only methylcytosine, the compound-converted DNA will not contain uracil in place of the methylcytosine.

[0024] The term “detection” is used herein to refer to any process of observing a marker, or a change in a marker (such as for example the change in the methylation state of the marker), in a biological sample, whether or not the marker or the change in the marker is actually detected. In other words, the act of probing a sample for a marker or a change in the marker, is a “detection” even if the marker is determined to be not present or below the level of sensitivity. Detection may be a quantitative, semi-quantitative or non-quantitative observation.

[0025] The term “differentially methylated nucleotide sequence” refers to a region of a genomic loci that is found to be methylated in advanced BE and / or cancer, but not methylated in the early stage of the disease.

[0026] The term “neoplasia” as used herein refers to an abnormal growth of tissue. As used herein, the term “neoplasia” may be used to refer to cancerous and non-cancerous tumors, as well as to Barrett's esophagus (which may also be referred to herein as a metaplasia) and Barrett's esophagus with dysplasia. In some embodiments, the Barrett's esophagus with dysplasia is Barrett's esophagus with high grade dysplasia. In some embodiments, the Barrett's esophagus with dysplasia is Barrett's esophagus with low grade dysplasia. In some embodiments, the neoplasia is a cancer (e.g., esophageal adenocarcinoma).

[0027] “Gastrointestinal neoplasia” refers to neoplasia of the upper and lower gastrointestinal tract. As commonly understood in the art, the upper gastrointestinal tract includes the esophagus, stomach, and duodenum; the lower gastrointestinal tract includes the remainder of the small intestine and all of the large intestine.

[0028] The terms “healthy”, “normal,” and “non-neoplastic” are used interchangeably herein to refer to a subject or particular cell or tissue that is devoid (at least to the limit of detection) of a disease condition, such as a neoplasia.

[0029] The term “including” is used herein to mean, and is used interchangeably with, theAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 phrase “including but not limited to.”

[0030] As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0031] As used herein, the term “event” refers to when a target biomarker is identified within a sample. “Copy number change” or “copy number alteration” (CNA) refers to any gain or loss of copies of section of DNA at somatic level. “Differential methylation” means a genomic region that has different methylation patterns among disease stages. As referred to herein, single nucleotide polymorphism or “SNPs” means changes in DNA sequence either in exon or intron regions, whereas “indel” means insertion / deletion polymorphism in the DNA or RNA sequence.

[0032] “Micro satellite instability” or MSI refers to change in length of repetitive DNA (show instability) suggesting fault in mismatch repair mechanism. Differential RNA expression or protein expression means over or under RNA or protein expression between early and late stage of the disease.

[0033] “Targeted enrichment or amplification” refers to enriching nucleic acid by means of any form of hybridization, PCR amplification, antibody pull down or enzymatic degradation of specific sequences.

[0034] The term “or” is used herein to mean, and is used interchangeably with, the term “and / or”, unless context clearly indicates otherwise.

[0035] A “sample” includes any material that is obtained or prepared for detection of a molecular marker or a change in a molecular marker such as for example the methylation state, or any material that is contacted with a detection reagent or detection device for the purpose of detecting a molecular marker or a change in the molecular marker.

[0036] As used herein, “obtaining a sample” includes directly retrieving a sample from a subject to be assayed, or directly retrieving a sample from a subject to be stored and assayed at a later time. Alternatively, a sample may be obtained via a second party. That is, a sample may be obtained via, e.g., shipment, from another individual who has retrieved the sample, or otherwise obtained the sample.Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24

[0037] A “subject” is any organism of interest, generally a mammalian subject, such as a mouse, and in particular embodiments, a human subject.

[0038] The terms “treat”, “treating”, and “treatment” as used herein, refer to both therapeutic treatment to prevent, either partially or completely, ameliorate, delay or slow down (lessen), inhibit (e.g., reducing or arresting the growth of) the targeted condition (e.g., EAC or BE), or one or more symptom associated therewith, or prolonging the life of a patient suffering from EAC. Subjects in need of treatment include subjects diagnosed with the disorder, subjects suspected of having the disorder, subjects predisposed to have the disorder. In some embodiments, treatment refers to the eradication, removal, modification, or control of cancer tissue or cancer cells. In some embodiments, treatment comprises administration of a therapeutic agent, including but not limited to proton therapy, immunotherapy, hormone therapy, toxin therapy or combination thereof and / or includes, but is not limited to chemotherapy, radiotherapy, phototherapy, cryotherapy, cryosurgery, toxin therapy high intensity focused ultrasound, dietary treatments, physical therapy or exercise regimens, surgical interventions, and combinations thereof.

[0039] As used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0040] Barrett's Esophagus (BE) is the precursor to EAC, with well-defined stages of disease progression. BE advances from Benign metaplasia, i.e., non-dysplastic BE (NDBE) to low-grade dysplasia (LGD), to neoplastic high-grade dysplasia (HGD), and finally to cancer.

[0041] Barrett's Esophagus is a condition in which there is an abnormal (metaplastic) change in the mucosal cells lining the lower portion of the esophagus. It is a major risk factor for EAC as most EAC is thought to develop from BE. Survival rate for EAC is below 25%. This is likely due to late-stage diagnosis. Traditional diagnosis methods can be prone to inaccuracy and lack of reproducibility. Screening for EAC generally involve endoscopic screening of patients with high-risk chronic gastroesophageal reflux disorder to determine the degree of dysplasia.

[0042] There is an unmet clinical need for improved methods for identifying patients at high risk and distinguishing between those that progress to EAC and those that do not.Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24

[0043] Aspects of the disclosure relate to compositions and methods for stratifying patients with BE. In some embodiments, the method comprises identifying patients who may have advanced neoplastic lesions, such as HGD or EAC. Such patients could benefit from immediate interventions, including but not limited to endoscopic eradication therapies or surgery, to reduce mortality. In some embodiments, the method comprises identifying patients who have not developed advanced neoplastic lesions (HGD or EAC). Such patients would continue to benefit from ongoing surveillance or monitoring at intervals defined by guidelines such as American Gastroenterological Association. For example, as intervals of from 1 year to 5 years, 2 years to 5 years, 3 years to 5 years, 4 years to 5 years, about every year, every 2 years, every 3 years, every 4 years, or every 5 years.

[0044] Aspects of the disclosure provide methods for diagnosing non-dysplastic BE (NDBE), low-grade dysplasia (LGD), neoplastic high-grade dysplasia (HGD), or esophageal adenocarcinoma (EAC) with a specificity or sensitivity that is greater than 70%. In some embodiments, the specificity and / or sensitivity of the method is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0045] In some embodiments, BE stages or EAC are identified with an accuracy of greater than 75%, 80%, 85%, 90%, 95%, 99% or more. In some embodiments, BE stages or EAC is identified with a sensitivity of greater than 95%. In some embodiments, BE stages or EAC is identified with a specificity of greater than 95%. In some embodiments, BE stages or EAC is identified with a sensitivity of greater than 95% and a specificity of greater than 95%. Biological sample

[0046] In some embodiments, the biological sample is a human biological sample. In some embodiments, the biological sample is an esophageal biological sample. In some embodiments, the biological sample is obtained by biopsy or any methods known in the art. In some embodiments, the biological sample is obtained by contacting the esophagus with a cytology brush or a balloon. In some embodiments, the biological sample is esophageal tissue sample or a sample comprising esophageal cells. In some embodiments, the esophageal cells of the tissue sample or cell sample are intact cells.

[0047] In some embodiments, the esophageal biological sample, such as but not limited to biopsy, cytology brushing or cytology sample, is collected using non-invasive cell sampling device such as EsoCheck, a cleared capsule balloon device.

[0048] The sample may be freshly obtained, formalin fixed, alcohol fixed, or paraffinAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 embedded.

[0049] In some embodiments, the biological samples are treated to extract nucleic acid, such DNA and RNA. In some embodiments, the nucleic acid, for example the DNA, is fragmented. In some embodiments, the DNA (or DNA fragments) is subjected to hybridization, such as hybridization capture, with an array of probes under conditions to allow hybridization. In some embodiments, the hybridized DNA (or DNA fragments) is bound to capture beads using methods known in the art. In some embodiments, the DNA (or DNA fragments) is subjected to an amplification reaction. In some embodiments, the amplified DNA (or DNA fragments) is sequenced. In some embodiments, the sequences are aligned to a reference human genome.

[0050] Software can be used to extract, normalize, summarize, and analyze data for each gene described herein. In some embodiments, the data in the biological sample are compared against a reference set to determine differential expression of one or more gene described herein. In some embodiments, the data in the biological sample are compared against a reference set to determine genetic alterations of one or more gene described herein. In some embodiments, the data, such as, but not limited to the number of copy and / or somatic mutation is normalized. For example, the data can be normalized using normal non-cancerous samples as baseline. In some embodiments, the copy number variation is determined. In some embodiments, the somatic mutation including allele frequency, depth of coverage, and expected impact is determined. Genetic alterations

[0051] In some embodiments, genetic alterations—such as copy number changes, single nucleotide polymorphisms (SNPs), and insertions / deletions (InDels), with or without epigenetic changes—are used as biomarkers to guide disease stratification. While previous technologies have attempted to achieve similar goals using, for example methylation changes in specific genes, or DNA alterations, such as aneuploidy interrogated at genome level, none of these technologies have interrogated copy number changes in defined genetic locations with or without other genetic (SNPs / Indels) and / or epigenetic alterations which can provide better differentiation ability.

[0052] In some embodiments, the difference in genetic alterations is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% or more. For example, the difference in gene copy number is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more. In some embodiments, the difference in geneticAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 alterations is at least 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more. For example, the difference in gene copy number is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 fold or more.

[0053] In some embodiments, disease stage is identified with an accuracy of greater than 75%, 80%, 85%, 90%, 95%, 99% or more. In some embodiments, disease stage is identified is identified with a sensitivity of greater than 95%. In some embodiments, disease stage is identified with a specificity of greater than 95%. In some embodiments, disease stage is identified with a sensitivity of greater than 95% and a specificity of greater than 95%.

[0054] In some embodiments, the biological sample is identified as cancerous with an accuracy of greater than 75%, 80%, 85%, 90%, 95%, 99% or more. In some embodiments, the biological sample is identified as cancerous with a sensitivity of greater than 95%. In some embodiments, the biological sample is identified as cancerous with a specificity of greater than 95%. In some embodiments, the biological sample is identified as cancerous with a sensitivity of greater than 95% and a specificity of greater than 95%. Methods

[0055] Methods of assessing a disease stage in a subject in need thereof are provided herein. In some embodiments, the method comprises classifying different BE stages and EAC. In some embodiments, methods of classifying early-stage BE (NDBE and LGD) from late-stage BE (HGD) and / or of classifying late-stage BE (HGD) from EAC are provided. In some embodiments, the method comprises determining a risk progression of BE in a subject.

[0056] Other aspects of the disclosure relate to methods of treating different BE stages and EAC.

[0057] In some embodiments, the method comprises the steps of: obtaining a biological sample comprising gene expression products; determining the expression level and / or genetic alterations for one or more gene expression products of the biological sample that are differentially expressed in BE / LGD stages and HGD / EAC stages; and identifying the biological sample as HGD / EAC wherein the gene expression level and / or genetic alterations for one or more gene expression products of the biological sample is indicative for HGD / EAC. In some embodiments, the gene expression level and / or genetic alterations for one or more gene expression products of the biological sample is indicative of the risk progression of BE (for example from BE / LGD HGD / EAC) in the subject. In some embodiments, the method further comprises treating the subject in need thereof. Treatment can include, but is not limited to, monitoring the patient and administering medication for stopping the production of stomach acid such as anti-acid medications including as proton pump inhibitors. Treatment can include,Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 but is not limited to, administering medications such as antacids, histamine blockers, and prokinetic agents. Treatment includes, but is not limited to, esophagectomy, chemotherapy, immunotherapies, radiofrequency ablation, and / or radiation therapy. Treatment can include, but is not limited to, administration of chemotherapeutic agents, inhibitors of growth factors, anti-angiogenic agents, antisense therapies, gene therapy, immunotherapy, etc. Treatment can include, but is not limited to, endoscopic ablation therapy, endoscopic photodynamic therapy, endoscopic cryotherapy, endoscopic mucosal resection, a surgical resection therapy, a non- endoscopic surgical therapy, or systemic therapy.

[0058] In some embodiments, the method comprises the steps of: obtaining a biological sample comprising gene expression products; determining the expression level and / or genetic alterations for one or more gene expression products of the biological sample that are differentially expressed in different BE stages and EAC; and identifying the biological sample as EAC wherein the gene expression level and / or genetic alterations for one or more gene expression products of the biological sample is indicative for EAC. In some embodiments, the method further comprises treating the subject in need thereof. Treatment includes, but is not limited to, esophagectomy, chemotherapy, immunotherapies and / or radiation therapy. Treatment can include, but is not limited to, administration of chemotherapeutic agents, inhibitors of growth factors, anti-angiogenic agents, antisense therapies, gene therapy, immunotherapy, etc. Treatment can include, but is not limited to, endoscopic ablation therapy, endoscopic photodynamic therapy, endoscopic cryotherapy, endoscopic mucosal resection, a surgical resection therapy, a non-endoscopic surgical therapy, or systemic therapy. In some embodiments, the treatment includes, but is not limited to, endoscopic mucosal ablation or esophageal resection to slow or prevent disease progression.

[0059] In some embodiments, the method comprises the steps of: obtaining a biological sample comprising gene expression products; determining the expression level and / or genetic alterations for one or more gene expression products of the biological sample that are differentially expressed in different BE stages and EAC; and identifying the biological sample as HGD wherein the gene expression level and / or genetic alterations for one or more gene expression products of the biological sample is indicative for HGD. In some embodiments, the method further comprises treating the subject in need thereof. In some embodiments, treatment includes, but is not limited to, endoscopic ablation, radiation frequency ablation, esophagectomy, chemotherapy, immunotherapies and / or radiation therapy. Treatment can include, but is not limited to, administration of chemotherapeutic agents, inhibitors of growthAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 factors, anti-angiogenic agents, antisense therapies, gene therapy, immunotherapy, etc. Treatment includes, but is not limited to, endoscopic mucosal ablation or esophageal resection to slow or prevent disease progression.

[0060] In some embodiments, the method comprises the steps of: obtaining a biological sample comprising gene expression products; determining the expression level and / or genetic alterations for one or more gene expression products of the biological sample that are differentially expressed in different BE stages and EAC; and identifying the biological sample as LGD wherein the gene expression level and / or genetic alterations for one or more gene expression products of the biological sample is indicative for LGD. In some embodiments, the method comprises repeating the method every about 6 months to 5 years, for example every about 6 months, year, 2 years, 3 years, 4 years, 5 years or more. In some embodiments, the method further comprises treating the subject in need thereof. Treatment can include, but is not limited to, monitoring the patient and administering medication for reducing stomach acid production. Treatment can include, but is not limited to, proton pump inhibitors, antacids or H2 blockers, prokinetic agents, photodynamic therapy and endoscopic mucosal resection.

[0061] In some embodiments, the method comprises the steps of: obtaining a biological sample comprising gene expression products; determining the expression level and / or genetic alterations for one or more gene expression products of the biological sample that are differentially expressed in different BE stages and EAC; and identifying the biological sample as NDBE wherein the gene expression level and / or genetic alterations for one or more gene expression products of the biological sample is indicative for NDBE. In some embodiments, the method comprises repeating the method every about 6 months to 5 years, for example every about 6 months, year, 2 years, 3 years, 4 years, 5 years or more. In some embodiments, the method further comprises treating the subject in need thereof. Treatment can include, but is not limited to, monitoring the patient, and administering medication. Treatment includes, but is not limited to, proton pump inhibitors, antacids or H2 blockers, photodynamic therapy and endoscopic mucosal resection.

[0062] In some embodiments, the biological sample is classified as EAC, HGD, LGD or NDBE with an accuracy of greater than 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%.

[0063] In some embodiments, a method for assessing a disease stage of a subject includes obtaining a sample from the subject, converting the sample to a modified sample, and analyzing the modified sample to identify a presence of at least one biomarker. In some embodiments,Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 the method comprises determining the disease stage of the subject. In some embodiments, the at least one biomarker comprises at least one of copy number alteration (CNA), differential methylation, single nucleotide polymorphisms / insertions-deletions (SNP / Indel), microsatellite instability (MSI), differential RNA expression, and changes in protein expression. In some embodiments, converting the sample comprises at least one of chemically, mechanically, or enzymatically converting the sample. In some embodiments, the at least one biomarker is differential methylation, and converting the sample includes converting the sample to differentiate methylated DNA bases from unmethylated DNA bases. In some embodiments, the at least one biomarker is CNA, and converting the sample includes at least one of reducing a size of DNA fragments within the sample and applying targeted enrichment of the sample. In some embodiments, the at least one biomarker is SNP / Indel, and converting the sample includes at least one of reducing a size of DNA fragments within the sample, applying targeted enrichment, and amplification of the sample. In some embodiments, the at least one biomarker is MSI, and converting the sample includes at least one of reducing a size of DNA fragments within the sample, applying targeted enrichment, and amplification of the sample. In some embodiments, the at least one biomarker is RNA expression or protein expression, and converting the sample includes at least one of reducing a size of DNA fragments within the sample, applying targeted enrichment, and amplification of the sample. In some embodiments, the disease stage qualifies the disease stage of Barrett’s Esophagus or esophageal cancer. In some embodiments, the method further comprises treating the subject.

[0064] Copy Number Alterations (CNA), RNA fusion, and / or single nucleotide polymorphisms / insertions-deletions (SNP / indels) can be determined using a panel encompassing all major DNA and RNA targets for cancer. In some embodiments, genetic alterations such as, but not limited to, CNA, RNA fusion and / or SNP / indels are determined using a genetic panel comprising one or more of the 311 genes of Table 1. In some embodiments, the genetic panel has 10 genes or more, 20 genes or more, 30 genes of more, 40 genes or more, 50 genes or more, 60 genes or more, 70 genes or more, 80 genes or more, 90 genes or more, 100 genes or more, 110 genes or more, 120 genes or more, 130 genes or more, 140 genes or more, 150 genes or more, 160 genes or more, 170 genes or more, 190 genes or more, 200 genes or more, 210 genes or more, 220 genes or more, 230 genes or more, 240 genes or more, 250 genes or more, 260 genes or more, 270 genes or more, 280 genes or more, 290 genes or more, 300 genes or more, or 310 genes of Table 1. In some embodiments, the genetic panel has from 50 genes to 311 genes of Table 1. In some embodiments, the genetic panel hasAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 from 60 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 70 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 80 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 90 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 100 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 120 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 140 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 160 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 180 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 200 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 200 genes to 220 genes of Table 1. In some embodiments, the genetic panel has from 220 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 240 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 260 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 280 genes to 311 genes of Table 1. In some embodiments, the genetic panel has from 300 genes to 311 genes of Table 1. Table 1 GeneHGNC No.TNFRSF1411912Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 PIK3C2B 8792 MDM4 6974Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 KLHL6 18653 EIF4A2 3284Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 ETV1 3490 INHBA 6066Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 RET 9967 ARID5B 17362Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 POLE 9177 LATS2 6515Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 GID4 28453 NF1 7765Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 NCOA3 7670 ZNF217 13009

[0065] In some embodiments, mean copy number alteration (number of copy number gains and losses within the sample) is higher in late-stage disease (HGD) and EAC than in early- stage disease (NDBE and LGD). In some embodiments, mean copy number alteration (number of copy number gains and losses within the sample) is higher in HGD / EAC than in NDBE or LGD. In some embodiments, the mean copy number alteration is from about 20 to about 100, from about 30 to about 100, from about 40 to about 100, from about 50 to about 100, from about 60 to about 100, from about 70 to about 100, from about 80 to about 100, from about 90 to about 100, from about 80 to about 90, from about 70 to about 80, from about 60 to about 70, from about 50 to about 60, from about 40 to about 50, from about 30 to about 40, from about 20 to about 30 higher in HGD / EAC than NDBE or LGD.

[0066] In another embodiment, a method for identifying a disease state of a sample from the subject includes obtaining the sample from the subject and analyzing the sample to determine a presence amount of at least one biomarker within the sample. In some embodiments, the method comprises determining the disease state of the sample through comparing a presence of an amount of the at least one biomarker with at least one predetermined value. In some embodiments, if the amount is above the at least one predetermined value, the disease state is late stage. In some embodiments, the at least one predetermined value is at least one value between twenty and ninety events, and if the presence amount is above the at least one predetermined value, the disease state is late stage. In further embodiments, the at least one predetermined value is forty-eight events, and if the presence amount is above forty-eight events, the disease is late stage. In some embodiments, the at least one predetermined value is at least one value between twenty and ninety events, and when the presence amount is less thanAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 or equal to the at least one predetermined value, the disease state is early stage. In further embodiments, the at least one predetermined value is forty-eight events, and if the presence amount is below forty-eight events, the disease is early stage. In some embodiments, the method further comprises treating the subject. In some embodiments, the event is the mean copy number alteration.

[0067] In other embodiments, a method for identifying a progression level of a disease of a sample includes obtaining the sample from a subject, converting the sample into a modified sample, measuring an amount of a target biomarker within the modified sample, the target biomarker comprising at least one of a copy number alteration (CNA) value, a single nucleotide polymorphisms / insertions-deletions (SNP / Indel) value, a microsatellite instability (MSI) value, a differential methylation value, a differential RNA expression value, and changes in protein expression, and comparing a value of the target biomarker with a predetermined value, wherein if the biomarker amount is below or equal to the predetermined value, a disease state of the sample is identified as early stage, and if the biomarker is above the predetermined value, the disease state of the sample is identified as late stage. In some embodiments, converting the sample into a modified sample includes treating the sample such that the DNA of the modified sample is fragmented and / or chemically modified. In further embodiments, the predetermined value is between twenty and ninety events. In some embodiments, the event is the mean copy number alteration. In some embodiments, the method further comprises treating the subject.

[0068] In other embodiments, a method for identifying a disease state of a sample from a subject includes obtaining the sample from the subject, analyzing the sample from the subject, analyzing the sample to quantify an amount of copy number alterations (CNA) within the sample, wherein the amount of copy number alterations is defined as the number of copy number gains and losses within the sample, and identifying whether the disease state of the sample is early stage or late stage. In some embodiments, the method further includes analyzing the sample to quantify a presence of gene mutations. In some embodiments, if the copy number gains and losses, and / or the number of mutations for one or more genes described herein is above at least one predetermined value, the disease state is late stage. In some embodiments, if the copy number gains and losses, and / or the number of mutations for one or more genes described herein is above at least one predetermined value, the disease state is late stage, and the method further comprises treating the subject. In some embodiments, if the copy number gains and losses and / or the number of mutations for one or more genes described herein is lower at least one predetermined value, the disease state is an earlier stage. In someAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 embodiments, if the copy number gains and losses and / or the number of mutations for one or more genes described herein is lower at least one predetermined value, the disease state is an earlier stage and the method further comprises repeating the method in 3-5 years, 1-3 years, 6 months to 1 year.

[0069] In some embodiments, the method comprises analyzing the sample to determine a mean copy number alterations and comparing the mean copy number alterations with at least one predetermined value. In some embodiments, the at least one predetermined value is at least one value between twenty and hundred mean copy number alterations. In some embodiments, the at least one predetermined value is about 90, and if the mean copy number alterations is above 90, the disease is HGD or EAC. In some embodiments, the at least one predetermined value is about 80, and if the mean copy number alterations is above 80, the disease is HGD or EAC. In some embodiments, the at least one predetermined value is about 50, and if the mean copy number alterations is above 50, the disease is HGD or EAC. In some embodiments, when the mean copy number alterations is less than or equal to about 20, the disease state is early stage (NDBE or LGD). In some embodiments, the method further comprises treating the subject.

[0070] Monitoring the levels of one or more biomarker such as copy number alternation (CNA) value, a single nucleotide polymorphisms / insertions-deletions (SNP / Indel) value, a microsatellite instability (MSI) value, a differential methylation value, a differential RNA expression value, and changes in protein expression can be used to monitor the progress of disease, such as before, during and / or after treatment. For example, levels or ratios of one or more biomarker of the disclosure being increased or decreased in subjects with EAC compared to a healthy control subject or a control subject with BE can be monitored. A change in the level or ratio of one or more biomarker in the subject to levels or ratios from control healthy subjects indicates that the disease has regressed, while change in the level or ratio of one or more biomarker in the subject to levels or ratios from in subjects having a more advanced stage of the disease indicates that the disease has progressed.

[0071] Methods for interrogating copy number changes in defined genetic locations and / or epigenetic alterations are provided herein.

[0072] In some embodiments, the method comprises hybridization capture to increase the coverage of interest by customized the probes at the regions of interest. Such method has the advantage to require lesser sequencing reads.Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24

[0073] Hybridization capture method provided herein has similar performance in comparison with commercial panel QIAseq® Multimodal Pan-Cancer Panel (Qiagen) comprising more than 520 genes in differentiating between advanced neoplastic lesions (HGD & EAC) and early-stage BE (NDBE and LGD).

[0074] Some aspects of the disclosure relate to a diagnostic test that can be utilized on a biological sample of a patient to stratify patients with BE.

[0075] Some aspects of the disclosure relate to genetic panels to stratify patients with BE. In some embodiments, the genetic panel comprises the genes of Table 1. In some embodiments, the genetic panel consists of the genes of Table 1. In some embodiments, the genetic panel comprises or consist of from about 50 gens to about 300 genes of Table 1 In some embodiments, the genetic panel comprises 10 or more genes of the genes of Table 1. In some embodiments, the genetic panel comprises or consists of 20 or more genes of the genes of Table 1. In some embodiments, the genetic panel comprises or consists of 30 or more genes of the genes of Table 1. In some embodiments, the genetic panel comprises or consists of 40 or more genes of the genes of Table 1. In some embodiments, the genetic panel comprises or consists of 50 or more genes of the genes of Table 1. In some embodiments, the genetic panel comprises or consists of 60 or more genes of the genes of Table 1. In some embodiments, the genetic panel comprises or consists of 70 or more genes of the genes of Table 1. In some embodiments, the genetic panel comprises or consists of 80 or more genes of the genes of Table 1. In some embodiments, the genetic panel comprises or consists of 90 or more genes of the genes of Table 1. In some embodiments, the genetic panel comprises or consists of 100 or more genes of the genes of Table 1. In some embodiments, the genetic panel comprises or consists of 150 or more genes of the genes of Table 1. In some embodiments, the genetic panel comprises or consists of 200 or more genes of the genes of Table 1. In some embodiments, the genetic panel comprises or consists of 250 or more genes of the genes of Table 1. In some embodiments, the genetic panel comprises or consists of 300 or more genes of the genes of Table 1.

[0076] In some embodiments, the genetic panel comprises or consists of 10 or more genes of the genes of Table 1, wherein the 10 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel comprises or consists of 20 or more genes of the genes of Table 1, wherein the 20 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel comprises or consists of 30 or more genes of the genes of Table 1, wherein the 30 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel comprises or consists of 40 or more genes ofAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 the genes of Table 1, wherein the 40 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel comprises or consists of 50 or more genes of the genes of Table 1, wherein the 50 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel comprises or consists of 60 or more genes of the genes of Table 1, wherein the 60 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel comprises or consists of 70 or more genes of the genes of Table 1, wherein the 70 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel comprises or consists of 80 or more genes of the genes of Table 1, wherein the 80 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel comprises or consists of 90 or more genes of the genes of Table 1, wherein the 90 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel comprises or consists of 100 or more genes of the genes of Table 1, wherein the 100 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel comprises or consists of 150 or more genes of the genes of Table 1, wherein the 150 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel comprises or consists of 200 or more genes of the genes of Table 1, wherein the 200 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel comprises or consists of 250 or more genes of the genes of Table 1, wherein the 250 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes. In some embodiments, the genetic panel comprises or consists of 300 or more genes of the genes of Table 1, wherein the 300 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes.

[0077] In some embodiments, the genetic panel comprises 10 or more genes of Table 1 and one or more additional gene that is not included in Table 1. Additional embodiments

[0078] Embodiment 1: A method for assessing a disease stage of a subject, the method including: obtaining a sample from the subject; converting the sample to a modified sample; analyzing the modified sample to identify a presence of at least one biomarker; and determining the disease stage of the subject.Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24

[0079] Embodiment 2: The method according to embodiment 1, wherein the at least one biomarker comprises at least one of copy number alternation (CNA), differential methylation, single nucleotide polymorphisms / insertions-deletions (SNP / Indel), microsatellite instability (MSI), differential RNA expression, and changes in protein expression.

[0080] Embodiment 3: The method according to embodiment 1, wherein converting the sample includes at least one of chemically, mechanically, or enzymatically converting the sample.

[0081] Embodiment 4: The method according to embodiment 3, wherein the at least one biomarker is differential methylation, and wherein converting the sample includes converting the sample to differentiate methylated DNA bases from unmethylated DNA bases.

[0082] Embodiment 5: The method according to embodiment 3, wherein the at least one biomarker is CNA, and converting the sample includes at least one of reducing a size of DNA fragments within the sample and applying targeted enrichment of the sample.

[0083] Embodiment 6: The method according to embodiment 3, wherein the at least one biomarker is SNP / Indel, and converting the sample includes at least one of reducing a size of DNA fragments within the sample, applying targeted enrichment, and amplification of the sample.

[0084] Embodiment 7: The method according to embodiment 3, wherein the at least one biomarker is MSI, and converting the sample includes at least one of reducing a size of DNA fragments within the sample, applying targeted enrichment, and amplification of the sample.

[0085] Embodiment 8: The method according to embodiment 3, wherein the at least one biomarker is RNA expression or protein expression, and converting the sample includes at least one of reducing a size of DNA fragments within the sample, applying targeted enrichment, and amplification of the sample.

[0086] Embodiment 9: The method according to embodiment 1, wherein the disease stage qualifies the disease stage of Barrett’s Esophagus or esophageal cancer.Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24

[0087] Embodiment 10: A method for identifying a disease state of a sample from a subject, the method comprising: obtaining the sample from the subject; analyzing the sample to determine a presence amount of at least one biomarker within the sample; and determining the disease state of the sample through comparing a presence of an amount of the at least one biomarker with at least one predetermined value.

[0088] Embodiment 11: The method according to embodiment 10, wherein the at least one predetermined value is at least one value between twenty and ninety events, and wherein if the presence amount is above the at least one predetermined value, the disease state is late stage.

[0089] Embodiment 12: The method according to embodiment 10, wherein the at least one predetermined value is forty-eight events, and wherein if the presence amount is above forty- eight events, the disease is late stage.

[0090] Embodiment 13: The method according to embodiment 10, wherein the at least one predetermined value is at least one value between twenty and ninety events, and when the presence amount is less than or equal to the at least one predetermined value, the disease state is early stage.

[0091] Embodiment 14: The method according to embodiment 10, wherein the at least one predetermined value is forty-eight events, and wherein if the presence amount is below forty- eight events, the disease is early stage.

[0092] Embodiment 15: A method for identifying a progression level of a disease of a sample, the method comprising: obtaining the sample from a subject; converting the sample into a modified sample; measuring an amount of a target biomarker within the modified sample, the target biomarker comprising at least one of a copy number alternation (CNA) value, a single nucleotide polymorphisms / insertions-deletions (SNP / Indel) value, a microsatellite instabilityAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 (MSI) value, a differential methylation value, a differential RNA expression value and changes in protein expression; and comparing a value of the target biomarker with a predetermined value wherein if the biomarker amount is below or equal to the predetermined value, a disease state of the sample is identified as early stage and if the biomarker is above the predetermined value, the disease state of the sample is identified as late stage.

[0093] Embodiment 16: The method according to embodiment 15, wherein converting the sample into a modified sample includes treating the sample such that unmethylated Cytosine bases are converted to Uracil bases.

[0094] Embodiment 17: The method according to embodiment 15, wherein the predetermined value is between twenty and ninety events.

[0095] Embodiment 18: A method for identifying a disease state of a sample from a subject, the method comprising: obtaining the sample from the subject; analyzing the sample to quantify an amount of copy number alterations (CNA) within the sample, wherein the amount of copy number alterations is defined as the number of copy number gains and losses within the sample; and identifying whether the disease state of the sample is early stage or late stage.

[0096] Embodiment 19: The method according to embodiment 18, wherein the method further includes analyzing the sample to quantify a presence of gene mutations.

[0097] The examples herein provide a method for identifying genetic, epigenetic, and / or proteomic biomarkers that can differentiate the early and late stages of disease.

[0098] The following examples are provided by way of illustration, not limitation. EXAMPLE Example 1: Genetic classifier to differentiate late-stage Barrett’s esophagus (HGD) and Esophageal adenocarcinoma (EAC) from early-stage disease (NDBE and LGD) Method

[0099] Fifty-nine Formalin Fixed Paraffin Embedded (FFPE) pinch biopsy samples wereAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 acquired from a biobank. Specimens consisted of 14 normal and 45 disease samples, including 23 NDBE, 7 LGD, 5 HGD, and 10 EAC. Reduced Representation Bisulfite Sequencing (RRBS) was performed on all 59 samples to detect differentially methylated regions. Additionally, Copy Number Alterations (CNA), RNA fusion, and single nucleotide polymorphisms / insertions- deletions (SNP / indels) were examined in all samples utilizing a large commercial panel (1.44 Mb) encompassing all major DNA and RNA targets for cancer. All data was analyzed for the ability to differentiate between NDBE and HGD & EAC. LGD was assessed either by grouping with NDBE, or with HGD & EAC due to known lack of reproducibility in LGD classification. LGD were ultimately grouped with early-stage based on molecular data. Results

[0100] Copy number gains. Losses and, gains and losses, were prevalent in late-stage BE (HGD) and cancer (EAC) in comparison to early-stage BE (NDBE and LGD), as illustrated in FIG.1.

[0101] In about 523 driver genes testing in the panel, TP53 showed the greatest number of somatic mutations impacting gene function related to HGD or EAC, as illustrated in FIG. 2. Wild-type TP53 gene is translated into p53 protein (p53) which is a transcription factor that acts in preventing the propagation of DNA-defective cells and the acquisition of cancer phenotype.

[0102] Copy number alterations (CNA) alone differentiate the early-stage BE (NDBE / LGD) from late-stage BE (HGD) and EAC between these samples with an area Under the Curve (AUC) of 0.896, with sensitivity at 73.3%, with specificity at 85.2%, and with accuracy at 81%, at a threshold of 47.5, as shown in FIG.3 and FIG.5.

[0103] Combining TP53 mutations with copy number alterations further improved predictive accuracy of the classifier while keeping a similar AUC. For example, copy number mutations and TP53 mutations could differentiate the early-stage BE from late-stage BE with an AUC of 0.899, with a sensitivity at 86.7%, with a specificity at 88.9% and with an accuracy at 88.1%, with a threshold of 36.5, as illustrated in FIG.4 and FIG.5. No differentially methylated region or RNA markers were identified that differentiated between disease stages.

[0104] A combination of genetic markers has the potential to be used as a classifier to differentiate among BE stages and EAC. The genetic classifier could potentially serve as a risk stratification tool to guide surveillance and / or treatment in patients with NDBE and LGD. Example 2: Genetic classifier to differentiate late-stage Barrett’s esophagus (HGD) andAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 Esophageal adenocarcinoma (EAC) from early-stage disease (NDBE and LGD) using 311 gene panel Method

[0105] Seventy (70) Formalin Fixed Paraffin Embedded (FFPE) pinch biopsy samples were acquired from a biobank. Specimens consisted of 21 normal and 49 disease samples, including 20 NDBE, 9 LGD, 15 HGD, and 5 EAC.

[0106] Target enrichment by hybridization capture was performed on all 70 samples. The data obtained after processing through bioinformatic pipeline are copy number variations and somatic mutations. All data was analyzed for the ability to differentiate between NDBE or LGD and HGD & EAC.

[0107] Due to the depth of coverage, no somatic mutation had a high impact on the stages' classifier on this specific set of samples. Results

[0108] fastq files from the sequencer were inputted in bioinformatic pipeline. The output of the bioinformatic pipeline is the copy number alteration, somatic mutation of each gene of the 311 genes in the panel of Table 1.

[0109] The bioinformatic workflow was a followed: 1. Demultiplex to separate individual signals for each gene for each sample. 2. Trim adapters. 3. Align the sequence to human reference genome (hg19). 4. Normalize the copy number using normal samples as baseline. 5. Determine copy number variation. 6. Determine somatic mutation including allele frequency, depth of coverage, and expected impact. Data analysis

[0110] The result for each sample was determined based on the total copy number variation and the impact of critical somatic mutation such as TP53.

[0111] The total number of copy number variation was the sum of the copy number gain and the copy number loss.

[0112] The copy number gain for each sample was determined by taking the sum of all the genes with copy number >2 assuming 2 copies is normal without gain or loss.Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24

[0113] The copy number loss for each sample was determined by taking the sum of all the genes with copy number <2.

[0114] The impact of somatic mutation is determined by the number of mutations of the specific gene per sample and number of COSMIC (Catalogue of Somatic Mutation in Cancers) mutations. The COSMIC mutations contribute a higher impact compared to non-COSMIC mutations. Results

[0115] The total copy number gains and losses could differentiate between early-stage BE (NDBE and LGD) and late-stage BE (HGD) and cancer (EAC).

[0116] Combining somatic mutations and copy number variations further improved the predictive accuracy of the assay.

[0117] An Area Under the Curve (AUC) and threshold was calculated based on the total copy number alteration and somatic mutation impact against the clinical diagnostic. The AUC was 86.5%. The AUC should be at least 80%.

[0118] The threshold was 32.79. The threshold can vary based on the normal baseline and the range is within 20 to 100, for example 20-60.

[0119] If the sample has a result less than the threshold (also referred herein as predetermined value), it was classified as early-stage BE.

[0120] If the sample has a result more than or equal to the threshold, it was classified as late- stage BE and cancer.

[0121] Analysis demonstrated that the sensitivity of the assay was 72% and the specificity was 89%.

[0122] Both the commercial panel and the panel of Table 1 exhibited a similar Area Under the Curve (AUC) showing 0.896 using commercial panel and 0.865 using custom panel when copy number alone were used as biomarkers. Thus, panel of Table 1 that captures specific genomic regions of interest has the potential to be used to identify HGD and EAC related changes in patients with BE. Example 3: Use in Diagnosis and Treatment Diagnosis

[0123] A method as described in Example 1 or 2 is used as a screening tool differentiateAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 between early-stage BE (NDBE and LGD) and late-stage BE (HGD) and cancer (EAC). Determination of EAC warrant referral to a specialty care provider for further evaluation and diagnosis. Treatment

[0124] Upon diagnosing early-stage BE (NDBE and LGD) and late-stage BE (HGD) and cancer (EAC) in a subject, the subject is treated for early-stage BE (NDBE and LGD) and late- stage BE (HGD) or cancer (EAC) using therapeutically effective treatments or using clinically accepted and / or approved treatments.

[0125] Upon diagnosing a subject with early-stage BE, treatment can include, but is not limited to, monitoring the subject and administering medications such as anti-acid medications, proton pump inhibitors, antacids, histamine blockers, and prokinetic agents. Treatment can include, but is not limited to, radiofrequency ablation, ablative therapy, and / or endoscopic eradication therapy. Upon diagnosing a subject with late-stage BE or EAC, treatment can include, but is not limited to, esophagectomy, chemotherapy, immunotherapies, radiofrequency ablation, and / or radiation therapy. Treatment can include, but is not limited to, endoscopic ablation therapy, endoscopic photodynamic therapy, endoscopic cryotherapy, endoscopic mucosal resection, a surgical resection therapy, a non- endoscopic surgical therapy, or systemic therapy.

[0126] The assay can be used to monitor the responsiveness of the subject to the treatment and / or to monitor the progress of the subject undergoing treatment.

[0127] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. It is understood that modifications which do not substantially affect the activity of the various embodiments of this disclosure are also provided within the description of the disclosure provided herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0128] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and / or theAttorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0129] Throughout this disclosure various publications, patents, and sequence database entries are mentioned. The disclosures of these publications, patents, and sequence database entries, including those items listed above, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0130] While the present disclosure has been described with reference to certain embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt to a particular situation, indication, material and composition of matter, process step or steps, without departing from the spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 CLAIMS What is claimed is:

1. A method for identifying a Barrett’s esophagus (BE) disease stage, the method comprising: (a) extracting nucleic acid molecules from an esophageal sample from a subject; (b) applying targeted nucleic acid sequence enrichment and sequencing the nucleic acid molecules; (c) quantifying an amount of copy number alterations (CNA) of fifty or more genes listed in Table 1, wherein the amount of CNA is defined as the number of copy number gains and losses within the nucleic acid, wherein if the amount is above a predetermined value, the disease stage is late stage of BE; and (d) identifying whether the disease stage is early stage or late stage of BE.

2. The method according to claim 1, the method comprising determining a CNA profile of the fifty of more genes listed in Table 1.

3. The method according to claim 1 or claim 2, wherein the fifty or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes.

4. The method according to any one of claims 1-3, wherein the method further comprises: quantifying presence of gene mutations of fifty or more genes listed in Table 1.

5. The method according to any one of claims 1-4, comprising quantifying presence of TP53 gene mutations.

6. The method according to any one of claims 1-5, wherein a significant increase in the CNA relative to the predetermined value is an indication that the subject has neoplastic high- grade dysplasia (HGD) or has esophageal adenocarcinoma (EAC).

7. The method according to any one of claims 1-6, wherein a mean CNA value over the predetermined value is an indication that the subject has neoplastic high-grade dysplasia (HGD) or has esophageal adenocarcinoma (EAC).

8. The method according to claim 7, wherein the predetermined value is greater than 20.

9. The method according to claim 7, wherein the predetermined value is from 20 to 100.

10. The method according to any one of claims 8-9, the method further comprising treating the subject.

11. The method according to claim 10, wherein the treating comprises removing at least part of the esophagus of the subject.

12. A method for monitoring Barrett’ s esophagus (BE) in a subject, the method comprising:Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 quantifying in a nucleic acid sample of the subject an amount of copy number alterations (CNA) of fifty or more genes listed in Table 1, wherein the amount of CNA is defined as the number of copy number gains and losses within the nucleic acid sample, wherein the subject has non-dysplastic BE (NDBE) to low-grade dysplasia (LGD) if mean CNA is lower than a predetermined value.

13. The method according to claim 12, wherein the predetermined value is about 20.

14. The method according to claim 12 or claim 13, further comprising monitoring BE in the subject.

15. The method according to claim 14 ,wherein the monitoring is every 3 to 5 years.

16. The method according to claim 14, comprising repeating the step of quantifying.

17. The method according to claim 14, comprising performing endoscopic surveillance.

18. A method of characterizing DNA is a biological sample of a subject, the method comprising: extracting DNA from the biological sample, wherein the biological sample comprises esophageal cells; fragmenting the DNA; constructing DNA fragment library; subjecting DNA fragments to hybridization capture; sequencing DNA fragments; aligning DNA sequences to reference human genome; determining mean CNA of fifty of more genes listed in Table 1; and using the mean CNA to determine if the DNA is derived from non-dysplastic BE (NDBE), low-grade dysplasia (LGD), neoplastic high-grade dysplasia (HGD), or Esophageal Adenocarcinoma (EAC) biological sample.

19. The method of claim 18, wherein the mean CNA over a predetermined value is an indication that the subject has neoplastic high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC).

20. A method of treating Barrett’s esophagus (BE) in a subject in need thereof, comprising: (a) obtaining a biological sample from the subject; (b) extracting nucleic acid from the biological sample; (c) analyzing the nucleic acid to quantify an amount of copy number alterations (CNA) of fifty or more genes listed in Table 1, wherein the amount of copy number alterations is defined as the number of copy number gains and losses;Attorney Docket No.: 155848-011802 / PCT Electronically Filed: 11-29-24 (d) determining whether the subject has early stage or late stage BE, wherein mean CNA over a predetermined value is an indication that the subject has late stage BE; and (e) monitoring or treating the subject according to the BE disease stage.

21. The method according to claim 19 or claim 20, wherein the predetermined value is greater than 20.

22. The method according to claim 19 or claim 20, wherein the predetermined value is from 20 to 100.

23. The method according to claim 22, wherein if the subject has non-dysplastic BE (NDBE) or low-grade dysplasia (LGD), the subject is monitored. method is repeated every 3 to 5 years.

24. The method according to claim 22, wherein if the subject has non-dysplastic BE (NDBE) or low-grade dysplasia (LGD), the method is repeated every 3 to 5 years.

25. The method according to claim 22, wherein if the subject has neoplastic high-grade dysplasia (HGD), proton pump inhibitors, antacids or H2 blockers, photodynamic therapy and endoscopic mucosal therapy.

26. The method according to claim 22, wherein if the subject has Esophageal Adenocarcinoma (EAC), the subjects is treated by endoscopic mucosal resection or removing at least part of the esophagus.

27. A genetic panel for diagnosing a subject with non-dysplastic BE (NDBE), low-grade dysplasia (LGD), neoplastic high-grade dysplasia (HGD), or Esophageal Adenocarcinoma (EAC), wherein the genetic panel comprises 50 or more genes of Table 1.

28. The genetic panel according to claim 27, wherein the genetic panel comprises from 50 to 311 genes of Table 1.

29. The genetic panel according to claim 27, wherein the 50 or more genes comprises MLH1, MSH2, MSH6 and PMS2 genes.

30. Use of the genetic panel according to any one of claims 27-29 to identify Barrett’s esophagus (BE) disease stage in a subject in need thereof.

31. Use of the genetic panel according to any one of claims 27-29 to stratify subjects with BE.

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