WO2018039422A1

WO2018039422A1 - Non-invasive detection of barrett's esophagus

Info

Publication number: WO2018039422A1
Application number: PCT/US2017/048357
Authority: WO
Inventors: M. Jonathan Bern
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-08-24
Filing date: 2017-08-24
Publication date: 2018-03-01
Anticipated expiration: 2019-02-24

Abstract

An office based non-invasive screening test for detection of Barrett's esophagus (BE) is disclosed. Over expression of specific molecular target molecules is used for the diagnosis of BE. In certain embodiments, the molecular targets are detected by quantitative reverse transcriptase PCR (RT-qPCR). In certain embodiments, a diagnostic test is provided that includes collection of esophageal brushings and testing for expression of one or more of MUC2 and CDX2, wherein expression of MUC2 and/or CDX2 in the esophagus is diagnostic for Barrett's esophagus. In certain embodiments, the diagnostic test may also utilize and immunoassay to determine MUC2 protein concentrations to establish the diagnosis of Barrett's esophagus.

Description

NON-INVASIVE DETECTION OF BARRETT'S ESOPHAGUS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority based on US Provisional Application Serial No. 62/379,048 filed 08/24/2016, and US Provisional Application Serial No. 62/430,059 filed 12/05/2016, each of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

[0002] This present disclosure relates generally to detection of precancerous conditions, especially Barrett's esophagus.

BACKGROUND OF THE INVENTION

[0003] Esophageal adenocarcinoma (EAC) is a major cause of cancer death in the United States and throughout the world. In 2014, there were 15,450 deaths from EAC in the United States alone. More disturbing is the increasing incidence of EAC. Over the past 20 years there has been an exponential increase in new EAC cases. Unfortunately, most EAC cases are only detected at a late stage when the patient presents with symptoms. EAC diagnosed at this advanced stage carries a very poor prognosis.

[0004] EAC begins as a precancerous lesion termed Barrett's esophagus (BE). BE represents a change from the normal squamous lining of the esophagus to a more acid resistant type of mucosa. This cellular transformation is called specialized intestinal metaplasia. The specialized intestinal metaplasia of BE is characterized by the development of mucin producing goblet cells. Thus, the current gold standard for diagnosis of BE in the United States is detection of goblet cell intestinal metaplasia (GCEVI) in biopsy samples. In England and in Europe, the presence of mucin producing goblet cells is not required for the diagnosis of BE.

[0005] The development of EAC from BE is a slow process proceeding though several stages as the cells lining the esophagus become progressively more abnormal. When seen by light microscopy these cellular changes are called dysplasia. Dysplasia progresses from low grade to high grade and eventually to EAC. Non-dysplastic BE caries a low risk of malignant transformation of 0.2-0.5% per year. The most recent data has shown that low grade dysplasia has an annual rate of progression to high grade dysplasia or EAC of 5.3% per year. High grade dysplasia has a significant rate of malignant transformation reported as 6.5 to up to 20% per year. Overall the risk of developing EAC in BE patients is increased at least 30-60 fold above that of the general population. [0006] If a nodule of dysplastic BE or early EAC is found, endoscopic mucosal resection (EMR) can be performed to remove the abnormal area of the esophagus. Early EAC can be cured with EMR. EMR is usually combined with a technique to ablate the full extent of BE. The preferred ablative technique is radiofrequency ablation (RFA) although balloon cryotherapy has also been used. EMR and RFA are well tolerated and are usually performed in an outpatient setting. Recent studies from Germany have shown that EMR treatment of early EAC results in a 92% - 96.3% five-year cancer free survival. This is a dramatic improvement compared to the current 18% five-year survival rate reported for EAC in the United States.

[0007] Because EAC starts as BE which is asymptomatic in at least 50% of patients, only screening will detect the majority of early curable cases. If BE with high grade dysplasia (HGD) or early EAC are identified by screening, minimally invasive endoscopic techniques offer an excellent chance for cure. Unfortunately, at this time most patients with EAC present with advanced disease, which carries a very poor prognosis. Screening of asymptomatic high- risk individuals is therefore necessary to reduce EAC mortality. While esophagogastroduodenoscopy (EGD) with biopsy is a very sensitive and specific diagnostic test for BE and EAC. EGD is expensive, mildly invasive and can only be performed by specialized providers such as gastroenterologists. As a result, routine screening of asymptomatic individuals with EGD is not recommended. Indeed, the American Gastroenterological Association (AGA) recommends only screening patients (with EGD) who have multiple risk factors associated with EAC for BE. Risk factors considered by the AGA include: age 50 years or older, male sex, white race, chronic GERD, hiatal hernia, an elevated body mass index and intra-abdominal distribution of body fat. Current surveillance guidelines for EAC in patients with known BE assume a 0.5% annual risk of cancer. The recommended surveillance interval is every 3-5 years looking for EAC or dysplastic BE.

[0008] From the foregoing, it appeared to the present inventors that an inexpensive highly accurate non-invasive office based screening test for BE could result in early detection of asymptomatic dysplastic BE and EAC and would save lives. Provided herein is the discovery of such an assay.

BRIEF SUMMARY OF THE INVENTION

[0009] Provided herein is an office based non-invasive screening test for detection of Barrett's esophagus (BE). In one embodiment, over expression of specific molecular target molecules is used for the diagnosis of BE. In one embodiment, the molecular targets are detected by quantitative reverse transcriptase PCR (RT-qPCR). In certain embodiments, molecular targets are CDX2, MUC2, MUC5UA, BMP4, villin and combinations thereof.

[0010] In certain embodiments, a diagnostic test is provided that includes collection of esophageal brushings and testing for expression of one or more of MUC2 and CDX2, wherein expression of MUC2 and/or CDX2 in the esophagus is diagnostic for Barrett's esophagus. (BE). In certain embodiment testing for expression of BMP4 is added.

[0011] In certain embodiments, a method for diagnosis of BE is provided including: a) obtaining a sample of esophageal cells from a subject; b) extracting nucleic acid from the sample; c) amplifying mRNA from the extracted nucleic acid using primers specific for expression from one or more of MUC2 and CDX2 genes; d) comparing an expression level of one or more MUC2 and CDX2 with expression levels of one or more housekeeping genes obtained from samples of cells from the subject to establish a gene expression level ratio for one or more of MUC2 and CDX2 in esophageal cells from the subject, e) determining that the ratio established in step d) is significantly different from expression of housekeeping gene(s) tested, and f) diagnosing said subject as having Barrett's esophagus. In certain embodiments, the expression of the genes is measured by a quantitative method selected from the group consisting of RT-qPCR, northern-blotting, microarray and SAGE.

[0012] In certain embodiments, collected esophageal cells are washed with a buffer such as phosphate buffered saline (PBS). The washed solution is assayed for MUC2 by enzyme linked immunosorbent assay (ELISA). In other embodiments, aliquots of collected cells are assayed for MUC2 and/or CDX2 by immunoassay. In certain embodiment testing for expression of BMP4 is added.

[0013] In certain embodiments, a method of diagnosing BE is provided inducing the steps of: (a) determining an expression profile of a predetermined set of mRNAs in an esophageal sample from a patient by an RNA reverse transcription reaction using mRNA-specific primers, and (b) comparing the expression profile of the predetermined set to a reference expression profile, wherein the comparison of the expression profile of the predetermined set with the reference set allows for diagnosis of Barrett's esophagus, and wherein the predetermined set of mRNAs diagnostic for Barrett's esophagus comprises at least MUC2 and CDX2 mRNA.

[0014] In certain embodiments, a patient suspected of having or being at risk for BE is tested by collection of esophageal cells by a tethered device that is swallowed and then recovered per orum via the tether. Cells from a surface lining of the esophagus are isolated from the device and mRNA is extracted from the cells. In certain embodiments, the device is a swallowed mesh sponge that is disposed in a gelatin capsule for swallowing and expands to fit against esophageal walls prior to being recovered.

BRIEF DESCRIPTION OF THE FIGURES

[0015] Fig. 1 shows results of RT-qPCR detection of TFF3 in the 15 patients detailed in Fig. 4.

[0016] Fig. 2 shows results of RT-qPCR detection of MUC2 in the 15 patients detailed in Fig. 4.

[0017] Fig. 3 shows results of RT-qPCR detection of MUC5AC in the 15 patients detailed in Fig. 4.

[0018] Fig. 4 shows the biographical and medical histories of the 15 patients tested in Fig. 1 - Fig. 3.

[0019] Fig. 5 graphs the mean MUC2 ACt in the two groups of patients segregated by the diagnosis of Barrett's Esophagus on pathology.

[0020] Fig. 6 graphs the mean CDX2 ACt in two groups of patients segregated by the diagnosis of Barrett's Esophagus on pathology.

[0021] Fig. 7 depicts a ROC curve of the sensitivity and specificity of use of MUC2 expression as a marker in the accurate diagnosis of Barrett's esophagus as confirmed by pathology.

[0022] Fig. 8 depicts a ROC curve of the sensitivity and specificity of use of CDX2 expression as a marker in the accurate diagnosis of Barrett's esophagus as confirmed by pathology.

[0023] Fig. 9A shows the RT-qPCR results for detection of MUC2 by brushings obtained from 17 patients with histologically confirmed BE compared to brushings from 23 patients with normal squamous epithelium. Fig. 9B shows the Delta delta Ct for MUC2 expression in BE brushings compared to normal squamous brushings.

[0024] Fig. 10A shows the RT-qPCR results for detection of CDX2 by brushings obtained from 17 patients with histologically confirmed BE compared to brushings from 23 patients with normal squamous epithelium. Fig. 10B shows the Delta delta Ct for CDX2 expression in BE brushings compared to normal squamous brushings. [0025] Fig. 11A shows the RT-qPCR results for detection of BMP4 by brushings obtained from 17 patients with histologically confirmed BE compared to brushings from 23 patients with normal squamous epithelium. Fig. 1 IB shows the Delta delta Ct for BMP4 expression in BE brushings compared to normal squamous brushings.

DETAILED DESCRIPTION OF THE INVENTION

[0026] Provided herein is an office based non-invasive screening test for detection of Barrett's esophagus (BE). In one embodiment, over expression of specific molecular target molecules is used for the diagnosis of BE. In one embodiment, the molecular targets are detected by quantitative reverse transcriptase PCR (RT-qPCR). In certain embodiments, molecular targets are CDX2, MUC2, BMP4, villin and combinations thereof.

[0027] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts which can be employed in a wide variety of specific contexts. The specific embodiment discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0028] ABBREVIATIONS: The following abbreviations are used throughout this application:

BE - Barrett's Esophagus

BMP4 - Bone Morphogenetic Protein 4

ΔΔΟΐ - delta delta cycle threshold or also referred to as Normalized Cycles to Threshold

CDX2 - Caudal Type Homeobox 2

EAC - Esophageal adenocarcinoma

EMT - epithelial mesenchymal transformation

EGD - esophagogastroduodenoscopy

GCIM - goblet cell intestinal metaplasia

GERD - gastroesophageal reflux disease

MUC2 - mucin subtype 2

MUC5AC - mucin subtype 5 AC

RT-qPCR - quantative reverse transcriptase polymerase chain reaction

TFF3 - Trefoil Factor 3

[0029] To facilitate the understanding of this invention, and for the avoidance of doubt in construing the claims herein, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. The terminology used to describe specific embodiments of the invention does not delimit the invention, except as outlined in the claims.

[0030] The terms such as "a," "an," and "the" are not intended to refer to a singular entity unless explicitly so defined, but include the general class of which a specific example may be used for illustration. The use of the terms "a" or "an" when used in conjunction with "comprising" in the claims and/or the specification may mean "one" but may also be consistent with "one or more," "at least one," and/or "one or more than one."

[0031] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives as mutually exclusive. Thus, unless otherwise stated, the term "or" in a group of alternati ves means "any one or combination of the members of the group. Further, unless explicitly indicated to refer to alternatives as mutually exclusive, the phrase "A, B, and/or C" means embodiments having element A alone, element B alone, element C alone, or any combination of A, B, and C taken together.

[0032] Similarly, for the avoidance of doubt and unless otherwise explicitly indicated to refer to alternatives as mutually exclusive, the phrase "at least one of when combined with a list of items, means a single item from the list or any combination of items in the list. For example, and unless otherwise defined, the phrase "at least one of A, B and C," means "at least one from the group A, B, C, or any combination of A, B and C." Thus, unless otherwise defined, the phrase requires one or more, and not necessarily not all, of the listed items.

[0033] The terms "comprising" (and any form thereof such as "comprise" and "comprises"), "having" (and any form thereof such as "have" and "has"), "including" (and any form thereof such as "includes" and "include") or "containing" (and any form thereof such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0034] Because BE represents a metaplastic change of the normal squamous esophagus, it results in several alterations in the cytopathology and molecular pathology of involved tissues. These include overproduction of two mucin species, mucin subtype 2 (MUC2) and mucin subtype 5AC (MUC5AC) and a marker for tissue metaplastic transformation, trefoil factor 3 (TFF3). See Hyland PL, et al. Global Changes in Gene Expression of Barrett's Esophagus Compared to Normal Squamous Esophagus and Gastric Cardia Tissues. PLoS ONE 9(4) (2014) e93219. Prior immunohistochemical studies of BE tissue biopsy materials have shown qualitatively elevated levels of MUC2, MUC5AC and TFF3. Overexpression of these protein markers has been shown to correlate with the likelihood of progression to HGD and EAC. Another study used a micro-hybidization array chip (chip type HG U133 A 2.0) to test for gene expression in fresh tissue (biopsy) obtained at EGD in BE and demonstrated a 69-fold increase in MUC5 AC compared to normal squamous mucosa.

[0035] However, certain prior markers and combinations thereof have found to be nonspecific. For example, a recent patent application (US 20120009597) discloses use of TFF3 as a marker for detection of BE in samples obtained with an esophageal cell sampling technique (CYTO SPONGE®). However, the present inventors determined that TFF3 does not specifically identify BE but is rather greatly influenced by inflammation making detection of TTF3 nonspecific and inappropriate for BE screening. The present inventors undertook to identify molecular markers for BE that were both specific for BE and sensitive enough to be detected in samples obtained non-invasively.

[0036] In one embodiment provided herein, the over expression of CDX2, MUC2, BMP4 and villin was found to be more sensitive and specific than the current standard of BE detection, EGD including narrow band imaging. CDX2 is a homeobox gene that encodes for an intestine- specific transcription factor, expressed in the nuclei of intestinal epithelial cells. The NCBI Reference sequence of Homo sapiens CDX2 mRNA is NM_001265. This target protein is noted throughout the intestine, from duodenum to rectum. As CDX-2 is a marker of epithelial intestinal differentiation it should not be present in esophageal or gastric mucosa under normal circumstances. When CDX-2 is seen in the esophagus it represents differentiation to intestinal epithelial cells. CDX-2 expression has been suggested as a marker for Barrett's esophagus when goblet cells cannot clearly be identified on histologic specimens. See Shi XY, et al. CDX2 and Villin Are Useful Markers of Intestinal Metaplasia in the Diagnosis of Barrett Esophagus. Am J Clin Pathol. 129(4) (2008) 571-7; Behan M, et al. Immunohistochemistry for CDX2 expression in non-goblet-cell Barrett's oesophagus. Br J Biomed Sci. 71(2) (2014) 86-92. Shi, XY et. al. showed that 100% of biopsies taken from patients with biopsy proven GCIM (as measured by the presence of alcian blue staining) stain positive for CDX2. Others, using laser capture microdissection to quantitatively measure CDX2 mRNA activity in reflux patients, found a stepwise increase in CDX2 gene expression starting with normal squamous epithelium (value of 1), followed by non-goblet intestinal metaplasia (NGFM) (40-70 fold increase), and GCFM (400-fold increase). Vallbohmer D, et al. Cdx2 expression in squamous and metaplastic columnar epithelia of the esophagus. Dis Esophagus Off J Int Soc Dis Esophagus ISDE. 19(4)(2006) 260-6.

[0037] The present inventors found, via immunohistochemical staining, that patients with GCIM and NGEVI demonstrated strong CDX2 immunohistochemical staining. CDX2 appears to be a potent marker for intestinalization of esophageal mucosa with and without goblet cells. In some cases, serial biopsies over several years have demonstrated loss of goblet cells but continued strong immunohistochemical staining for CDX2. This indicates continued intestinalization of the esophageal mucosa in the absence of goblet cells. Thus, the present inventors believe that BE is still present in patients despite the loss of characteristic goblet cells and that these patients continue to be at risk for esophageal cancer.

[0038] BMP-4 belongs to the transforming growth factor β family (TGF-β) and has multiple roles in cell proliferation, differentiation and cell migration. Bone morphogenetic proteins (BMPs) consist of more than 20 family members each having their own BMP-receptor affinity and regulating ligands. BMPs bind to transmembrane serine-threonine kinase receptors type 1 and 2 and activation of this leads to phosphorylation of SMAD1, 5 and 8, which permits binding to SMAD4. This complex translocates to the nucleus and regulates gene transcription specific for the BMP pathway. In the past BMP's were primarily felt to function during embryogenesis, however recent studies have shown that BMP's are overexpressed in various malignancies and may play a major role in cancer development. BMP-4 has been shown to be significantly upregulated BE and EAC.

[0039] Very recently it was shown that when BMP-4 is applied to a BE cell culture line, epithelial mesenchymal transformation (EMT) is induced. See Kestens C, et al. BMP4 Signaling Is Able to Induce an Epithelial -Mesenchymal Transition-Like Phenotype in Barrett' s Esophagus and Esophageal Adenocarcinoma through Induction of SNAIL2. PLoS ONE 11(5) 2016 May 18. This indicates a major role for BMP-4 in the pathogenesis of BE and EAC. Overexpression of this molecular target in esophageal tissue not only serves as a marker for BE but also gives a window of observation into the pathogenesis of BE and EAC.

[0040] Mucin 2 (MUC2) is particularly prominent in the gut where it is secreted from goblet cells lining the lumen of the large intestine. The NCBI mRNA Reference Sequence is M_002457.4. In the large intestine MUC2, along with other mucin proteins, polymerizes to form a gel. This gel provides an insoluble mucous barrier that protects the intestinal epithelium. MUC2 should not be present in the esophagus or stomach under normal circumstances. Studies have shown that MUC2 serves as a marker for GCIM and NGIM. See e.g. Chinyama CN, et al. Expression of MUC1 and MUC2 mucin gene products in Barrett's metaplasia, dysplasia and adenocarcinoma: an immunopathological study with clinical correlation. Histopathology 35(6) (1999) 517-24. Our studies support these findings and indicates this to be a potent target molecule for a BE test.

[0041] Villin is a tissue-specific actin-binding protein associated with the actin core bundle of the brush border. Villin is believed to function in bundling, nucleation, capping and severing of actin filaments. Villin proteins help to support the microfilaments of the microvilli on the brush border of the intestine. This is an intestine specific protein and should not be found in the esophagus or stomach under normal circumstances and therefore may serve as a marker for BE. Shi XY et.al. {supra) found that 100% of biopsy with proven GCIM had villin by immunohistochemical staining.

[0042] In one embodiment, patients are tested at the time of routine EGD. In certain embodiments, the test is applied to patients that fall in to high risk groups for BE even if they are not overtly symptomatic. BE is usually discovered during endoscopic examinations of middle-aged and older adults with symptomatic gastroesophageal reflux disease (GERD). The mean age at the time of diagnosis is 55 years, with the highest prevalence rates noted for white and Hispanic men. Men are much more likely to develop BE compared to women with an odds ratio of 2.6 to one. Asians and African Americans have significantly lower BE rates. Among patients who have endoscopic examinations because of symptomatic chronic GERD, long-segment BE (greater than 3 cm in length) is found in 3 to 5 percent of the patients, whereas 10 to 15 percent have short-segment BE.

[0043] A recent evaluation using the National Cancer Institute SEER data base suggests a high prevalence rate for BE in the United States at 5.6%. A recent study from Olmstead county Minnesota (Mayo Clinic) supports this estimate in a prospective study that found 4.4% prevalence of BE in the general population. This study comprised 1157 randomly selected persons that underwent transnasal endoscopy. The study population was 58% women and 42% men and therefore may have underestimated the true prevalence. Sami, S.S., Dunagan, K.T., et. al. A Randomized Comparative Effectiveness Trial of Novel Endoscopic Techniques and Approaches for Barrett's Esophagus Screening in the Community. Am. J. Gastroenterol. 110 (2015) 148-158. [0044] Applying this prevalence data to the United States population would translate to about 21 million individuals with BE.

[0045] Clearly GERD is a major risk factor for BE and EAC. A meta-analysis of five case- control studies with 1189 patients with EAC and 4666 controls found that patients with occasional GERD symptoms were more likely to develop EAC than those patients with no GERD symptoms (odds ratio (OR) 4.9) and the risk of developing esophageal adenocarcinoma is much higher in patients with daily GERD symptoms (OR 7.4).

[0046] Obesity is a risk factor for both GERD and BE. A 2009 meta-analysis that included 11 observational studies demonstrated an increased risk of BE in patients with a body mass index (BMI) >30 kg/m² as compared with patients with a BMI <30 kg/m² (OR 1.4). Other studies have shown that central obesity is an even more robust predictor of BE. Central obesity in these studies is defined as a high waist to hip ratio (>0.9 in males and >0.85 in females). Subjects with BE were also significantly more likely to currently or previously smoke or had ever smoked cigarettes compared to the general population (OR 1.67). Increasing pack-years of smoking correlates with increased the risk for BE.

[0047] In certain embodiments, the test is applied as a routine non-invasive office based test for a patient subset considered to be of increased risk for BE. In one embodiment the patient subset is adult male patients over the age of 18 with intermittent or chronic GERD symptoms. In other embodiments, the patient subset includes individuals with known BE or previously treated BE that is being seen for surveillance endoscopy. In certain embodiments, the biomarker testing is conducted on asymptomatic patients in the highest risk population. In one embodiment, testing is performed in middle aged (50 years and older) white or Hispanic men with central obesity having occasional or no GERD symptoms. This targeted approach is directed to capture the bulk of the at-risk population for EAC. If a positive score is obtained with one or more of the biomarker subset comprising MUC2, BMP4 and villin, EGD with biopsy would be the next step. If high grade dysplastic (HGD) BE or early EAC were identified, EMR and RFA could be performed with a high chance for cure. If BE with low grade dysplasia (LGD) were identified, RFA alone could be performed with a high chance of cure. If non-dysplastic BE was identified, surveillance EGD could be performed looking for development of dysplasia or EAC and treatment. All patients with BE should be treated with a proton pump inhibitor to reduce the risk of malignant progression. See Singh, S., Garg, S.K., et. al. Acid-suppressive medications and risk of oesophageal adenocarcinoma in patients with Barrett's oesophagus: a systematic review and meta-analysis. Gut 63 (2014) 1229-1237. Current screening guidelines for non-dysplastic BE would call for repeat EGD every 3-5 years.

[0048] Cells are sampled by a method that collects esophageal cells by a minimally invasive scraping or brushing procedure with a retrievable collection device that is swallowed. In one embodiment, the cells are collected with a cytological brush technique. In one embodiment, a tethered sponge disposed in a gelatin capsule is swallowed. When the capsule dissolves in the stomach and the sponge is released and withdrawn by the tether. Esophageal brushings are tested by a method that measures the expression level of normal and marker genes in the esophagus by determining the amount of mRNA or of the cDNA corresponding to the genes. Preferably the method is quantitative method. Another method may utilize a predetermined cut off point separating a positive from negative result producing a qualitative result. One such method for measuring relative amounts of mRNA expression is reverse transcription quantitative polymerase chain reaction (RT-PCR followed with qPCR). RT-PCR initially generates a complementary DNA (cDNA template from the mRNA by reverse transcription. The cDNA template is then used for qPCR where fluorescence of a probe changes as the DNA amplification progresses. Using a standard curve, qPCR is able to quantitate the relative levels of mRNA species within the sample. Another embodiment utilizes direct measurement of MUC2 and/or CDX2 protein levels by immunoassay on collected cells or from a supernatant solution of collected cells.

[0049] Levels of mRNA can also be quantitatively measured by other several methods including northern blotting which gives size and sequence information about the mRNA molecules including discrimination of alternately spliced transcripts. Other methods known in the art include use of DNA microarrays and techniques such as Serial Analysis of Gene Expression (SAGE), which provides relative measures of different mRNAs.

[0050] As exemplified herein, RT-qPCR was selected to evaluate relative expression of selected markers from cells obtained by esophageal brushing. Esophageal brushings are minimally invasive and represent little or no risk to the patient.

[0051] In certain embodiments, brushings are obtained from two locations for each patient: the suspected site of BE and normal squamous mucosa as an internal control. In certain embodiments, two to four passes of the cytological brush are performed from each site and the brush is cut from the endoscopic appliance and placed in specimen containers. The tissue and brush samples from each of the sites is placed RNA sample collection tubes such as for example RNAlater TissueProtect Tubes (Qiagen) in order to preserve the integrity of the RNA in the samples and then sent to the lab for immediate processing and storage at -20°C. When all the specimens have been collected, RT-qPCR analyses is performed batch wise. This batch assay method minimizes variation between assays.

[0052] In certain embodiments, a kit is provided for use in identifying patients for whom further work-up is required to confirm BE or to establish a surveillance schedule. In certain embodiments, the kit includes a swallowable tethered collection device in a sterile wrapper and an RNA sample collection tube including sample buffer wherein the tube includes a tag with identifies the patient and directs testing by Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) for one or more of MUC2 and CDX2 together with testing of housekeeping gene expression. The kit is prepared for storage and transport to a testing facility that processed the sample in accordance with the directed testing.

[0053] RT-qPCR is sufficiently sensitive to detect and quantify very small amounts of target mRNA and therefore look for overproduction of specific proteins. RT-qPCR uses gene amplification and therefore can detect very low mRNA levels. RT-qPCR assays employ fluorescent reporter probes (i.e. TaqMan, Life Technologies) that can be designed for specific mRNA targets which results in minimal cross reactivity and high specificity. In certain embodiments, the RT-qPCR assays employed have a 98% sensitivity over a 7-log concentration range with a gene amplification efficiency of over 98%.

[0054] Total RNA is isolated using standard protocols such as for example using the RNeasy mini kit with gDNA eliminator column (Qiagen Inc.). Total mRNA from each sample is converted to complementary DNA (cDNA) by available methods such as for example using the High Capacity RNA to cDNA kit (Life Technologies). Depending on the estimated numbers of calls collected, the cDNA may undergo preamplification such as for example using the TaqMan PreAmp Master Mix Kit (Invitrogen) prior to qPCR to amplify the cDNA targets equally without introducing bias while increasing the amount of total cDNA that may be required when assaying for multiple targets. In one embodiment, MUC2, CDX2, BMP4 and villin mRNA expression levels are assessed using TaqMan Gene Expression Assay kits specific for these target proteins (Life Technologies). Over expression of the target genes may be expressed in a number of ways including as an aggregate score of target gene expression over control gene expression or as over expression of an individual target gene. Target gene overexpression may also be expressed in absolute terms such as copies per microliter. [0055] In certain embodiments, the RT-qPCR data is expressed in "Cycles to Threshold" (Ct). As understood by those of skill in the art, Ct is a relative value representing the cycle number at which the fluorescent signals of the amplified DNA reaches a determined threshold level that exceeds background. Because of inter-assay variability and differences in cell numbers from which mRNA is extracted, Ct values are typically normalized against Ct amplification values for a constitutively expressed reference sequence such as a housekeeping gene. Thus, differential expression is considered gene by gene and expressed as normalized Ct values (ACt) of biological replicates between groups of samples. As stated herein, the normalized CT values are also referred to as AACt. In resulting expression, Ct levels are inversely proportional to the amount of target nucleic acid in the sample such that high ACt values represent low expression of a given gene while highly expressed genes have low ACt values. See e.g. Goni, R, et al. The qPCT Data Statistical Analysis. Integromics White Paper, September 2009, available on-line.

[0056] If desired, biopsies of suspected BE are obtained and sent for histologic evaluation by a gastrointestinal histopathologist. Such histology has historically served as the gold standard for the diagnosis of BE. In certain embodiments, in addition to standard histology techniques, immunohistochemical stains are also examined for MUC2, CDX2, BMP -4 and villin. The RT- qPCR mRNA expression levels for the target proteins can be compared between suspected BE and normal squamous mucosa within each patient.

[0057] In certain embodiments, an office based screening test is provided to detect intestinalization of the esophagus, also known as intestinal metaplasia or Barrett's esophagus (BE). The test will use a non-invasive cell sampling technique to obtain esophageal mucosal cells for analysis. In certain embodiments, the cell sampling technique employs a course mesh collection device. One non-limiting example of a suitable device is the CYTOSPONGE® (Medtronic GI Solutions), which is a single-use device used to collect cells from the lining of the esophagus. It consists of a small mesh sponge, about 30 mm in diameter, contained in a gelatin capsule and attached to a string. The patient swallows the capsule with water and the gelatin coating dissolves once the device reaches the stomach. After approximately five minutes, the string is used to retrieve the expanded sponge. As it is retrieved, the slightly abrasive mesh collects cells along the length of the esophagus. The cell sampling device is placed in a buffer such as phosphate buffered saline (PBS) and agitated to free the cells from the sponge. The solution containing esophageal cells is centrifuged to form a cellular pellet. The cellular pellet is placed in the RNA preservative. Preferably the RNA preservative shall maintain the integrity of the RNA sample for at least 7 days at room temperature to allow transport and processing at a central laboratory facility. Over expression of the target genes may be expressed as an aggregate score of target gene over expression or as over expression of an individual target gene.

[0058] In certain embodiments, a positive test is followed by EGD to confirm the diagnosis of BE and exclude dysplasia or EAC. If dysplastic BE or early EAC are identified, ablative techniques can be used to treat these conditions to prevent progression to invasive EAC. The preferred ablative techniques are EMR and RFA although other techniques may be used depending on local expertise.

[0059] In other embodiments, methods and kits are provided that detect expression of one or more of MUC2, MU5AC, CDX2, BMP4 and villin, either directly or by detection of a corresponding mRNA and/or cDNA, including be including by quantitative reverse transcription polymerase chain reaction (RT-qPCR). In some instances, an increase in expression of one of these proteins (or corresponding mRNA or DNA) is detected. In other instances, a decrease in expression of one of these proteins (or corresponding mRNA or DNA) is detected.

[0060] Detection of protein may be accomplished using numerous means including immunoassays such as enzyme linked immunosorbent assays (ELISA), using agglutination assays to measure protein content, radio-immunoassays, and using mass spectrometry to detect one or more characteristic fragment peaks of the proteins or by other methods. In one embodiment, the MUC2 and/or CDX2 concentration is determined by immunoassay of wash and/or carrier solutions. In other embodiments, aliquots of collected cells are tested for MUC2 and/or CDX2 and/or BMP4 expression by immunoassay.

[0061] In an embodiment, the cell sampling device containing collected esophageal cells is placed in a buffer such as phosphate buffered saline (PBS) and agitated to free the cells from the sponge. At least a portion of the solution containing esophageal cells is used to assay for cellular expression of MUC2 and/or CDX2 by immunoassay, including by ELISA. In one embodiment, the cell sample is allowed to rest at room temperature or at 37° for a period of time such as for example approximately 10 minutes to approximately 30 minutes. The sample is centrifuged and an aliquot of the supernatant is removed for testing by immunoassay for MUC2 elaborated by the cells. In still other embodiments, whole cells from the cell sample are assayed by immunoassay such as an ELISPOT assay using mesh filter plates in 96 well format and adapted to use on a vacuum manifold for washing.

[0062] In other embodiments, the cell sample is centrifuged to collect a cell pellet. The cells in the pellet are resuspended in a small volume and lysed such as by freeze thawing. Cellular debris is removed such as by centrifugation and the cell lysate is testing by immunoassay for expressed levels of MUC2, CDX2 and BMP4. For each of these immunoassays, target specific antibodies directed to the target protein are utilized. Depending on the assay, binding detection may be obtained by enzyme or fluorescent labelled polyclonal detection antibodies. Anti- human MUC2 antibodies may be obtained as murine monoclonals or rabbit polyconal antibodies from LSBio among others.

[0063] The methods and kits provide for non-invasive collection and may use a sponge, e.g., a capsule sponge, or other material that can be swallowable to collect cells along the esophagus by scraping or brushing. The exact nature of the sponge may vary, but in some instances, the sponge is cellulose based to permit digestion of the cellulose with an enzyme, e.g., a cellulase, and liberation of any cells within or on the sponge. The cells can be lysed to release one or more of MUC2, MU5 AC, CDX-2, BMP -4 and villin proteins and any mRNA corresponding to the proteins. Primers specific for one or more of MUC2, MU5AC, CDX2, BMP4 and villin are used to reverse transcribe the mRNA into a cDNA. The cDNA levels are detected and used to determine if the subject is at risk for Barrett's Esophagus, has Barrett's Esophagus or has EAC . The sponge can be coupled to a string or other device such that after the subj ect swallows the sponge, the sponge can be retrieved by pulling on the string. Suitable primers and enzymes are commercially obtainable from Invitrogen to permit RT-qPCR of the mRNAs that encode one or more of MUC2, MU5AC, CDX2, BMP4 and villin.

[0064] The methods and kits may also be non-endoscopic. In some instances, the kit may be provided as an over the counter kit where the subject swallows the sponge to permit cell sampling of the esophagus. The subject may then extract the sponge and place the sponge, e.g. after cutting of the string if desired, into a suitable buffer solution, e.g., phosphate buffered saline, or other solution to stabilize the cells or the mRNA species in the cells. The sponge + buffer solution is shipped to a laboratory for analysis. The solution may include cellulose or other enzymes if desired. In some instances, the solution may comprise detergents, salts, etc. to stabilize the cells or, if desired, lyse the cells. The sponge can be shipped in dry ice or at a reduced temperature to reduce the likelihood of mRNA degradation. EXAMPLE 1

RT-qPCR testing for TTF3, MUC2 and MU5AC.

[0065] Esophageal brushings were obtained at the time of EGD from 15 patients detailed in Fig. 4. Two female and 13 male patients were evaluated of which ten patients had a prior history of BE. Endoscopic diagnosis and biopsy evaluations showed either Short Segment BE (SSBE) or Long Segment BE (LS BE) in 13 patients while one received a normal endoscopic diagnosis. Three passes of the cytological brush were conducted and the brush cut from the endoscopic appliance and placed in RNAlater TissueProtect Tubes (Qiagen) and then sent to the lab for immediate processing and storage at -20°C. Total RNA was isolated using the RNeasy mini kit with gDNA eliminator column (Qiagen Inc.). Total mRNA from each sample was converted to cDNA using the High Capacity RNA to cDNA kit (Life Technologies). The cDNA was preamplified using the TaqMan PreAmp Master Mix Kit (Invitrogen division of ThermoFisher) prior to qPCR in order to amplify the cDNA targets. TTF3, MUC2, MU5AC, CDX2, BMP4 primers were used for amplification using TaqMan Gene Expression Assay kits (InVitrogen division of ThermoFisher, Cat. #4331182). The selected targets and amplicons are set out in Table 1.

[0066] Table 1: Gene Expression Targets and Amplicons selected

* Housekeeping gene, Human actin beta, mRNA

[0067] Data on TTF3 expression is presented in Fig. 1, data on MUC2 expression is presented in Fig. 2 and data on MUC5 AC is presented in Fig. 3. In each case meaningful data was obtain using RTqPCR to determine levels of marker expression in samples collected by a retrievable cytological collection device. However, only a subset of the tested molecular targets, TFF3, MUC2, MUC5AC, were determined to be selective for BE based on subsequent analysis.

[0068] Our preliminary data (as presented in the tables and graphs of Figs. 1 - 4) showed that TFF3 results by RT-qPCR were much more influenced by inflammation as seen on biopsies rather than to the presence of BE. Thus TTF3 does not appear to be a specific marker for BE and is probably not appropriate in isolation for a BE screening test. This result contrasts with the results disclosed in US 20120009597, which discloses use of TFF3 as a marker for detection of BE in samples obtained with an esophageal cell sampling technique (CYTOSPONGE®). Notably, in the application published as US 20120009597, immunohistochemical staining was used for detection of TFF3 in lieu of RT-PCR.

[0069] MUC2 was shown to be a viable marker for a BE screening test. It is normally only seen in the colon and is produced by mucin producing goblet cells. This marker should not be seen in the stomach or esophagus but is found in these locations in BE. Our preliminary results show significant overexpression of MUC-2 in BE and this correlates with histology for BE. This was true for non-goblet intestinalization (NGIM) and goblet cell intestinalization (GCIM).

[0070] CDX2 is a marker of intestinalization. Our lab and others have found strong immunohistochemical staining in NGIM, GCIM and esophageal adenocarcinoma (EAC). This marker should not be found in the stomach or esophagus under normal circumstances. This is currently used in most advanced clinical pathology labs to confirm intestinal origin for tumors. The pathology findings from our study has resulted in routine use of immunohistochemical staining for CDX2 in suspected BE cases at our institution.

[0071] BMP4 is seen in BE and EAC by immunohistochemical staining and may relate to the pathogenesis of EAC.

[0072] In certain embodiments, a positive result by RT-qPCR for any of CDX2, MUC2 and BMP4 in samples collected with a non-invasive retrievable cytological collection device will stage the patient for further evaluation.

[0073] Villin expression tracks along with CDX2 but is another marker that is expected to be useful in the context of results for additional markers. The immunohistochemical findings are a bit less overt under the microscope but the RT-qPCR result may correlate better with BE by histology. [0074] MUC5AC was chosen based on review of the literature at the time. However, MUC5AC is also seen in the cardia of the stomach making this nonspecific for BE when considered in isolation. On the other hand, our data does show that MUC5AC correlates with BE in our studies and tracks along with MUC2. While this marker may not be specific enough for a BE screening test if considered in isolation, it remains a viable candidate marker when used together with other markers.

EXAMPLE 2

Expanded cohort RT-qPCR testing for MUC2, CDX2 and BMP4.

[0075] An expanded cohort of patients was tested by RT-qPCR to hone in on a combination of assays that would provide 100% sensitivity with high specificity. As defined herein high specificity meets or exceeds 60%. Samples were collected and processed as disclosed in Example 1. The patients tested in Example 1 were included with further patients and data was collected for expression of MUC2, CDX2 and BMP4. The demographics of the patient cohort are shown in Table 2.

Table 2: Cohort Demographics and Data

[0076] Of the cohort of 40 patients, 33 were male (82.5%). Thirty eight out of the 40 patients (95%) were considered positive for Barrett's by esophagogastroduodenoscopy (EGD). EGD examinations were performed with a high definition Olympus upper endoscope equipped with narrow band imaging (NBI). All EGD exams were performed by an experienced attending gastroenterologist. A specialty trained GI histopathologist interpreted all pathology specimens. However, only 17 patients (42.5) were confirmed to have Barrett' s esophagus by pathology including detection of the presence of goblet cells in the esophagus, while 23 patients were confirmed to not have Barrett' s. Thus, EGD, despite being invasive and expensive, has relatively poor predictive value for the diagnosis of BE based on endoscopy findings when compared to histopathology by an expert GI histopathologist including the findings of goblet cells (the gold standard).

[0077] Based on the original results showing lack of BE specificity for TTF3 and MUC5AC, testing for these markers was not continued by RT-qPCR in the expanded cohort. All esophageal brushings were obtained at the time of EGD using a cytology brush and were tested for MUC2, CDX2 and BMP4 by RT-qPCR as described in Example 1. The results are presented in Table 2. The MUC2 AACt, CDX2 AACt and BMP4 AACt columns present the data normalized to a reference or control signal of constitutively expressed mRNA. The control represents RT-qPCR data from esophageal brushings taken from the distal esophagus of the patient that proved not to have BE by histology but looked like BE at the time of EGD. Table 3 shows an analysis of the AACt data together with verified detection of Barrett's by pathology.

Table 3: Mean AACt of the variables based on pathology result

[0078] As can be seen on Table 3, both MUC2 and CDX2 have considerably lower AACt values in patients who have had Barrett's esophagus confirmed by pathology. Even given the small sample population, the ratio of expression (AACt) of CDX2 to control genes was 1.476. For MUC2, the ratio of expression (AACt) of MUC2 to control genes was 3.33.

[0079] Fig. 5 graphs the mean MUC2 AACt in the two groups of patients segregated by the diagnosis of Barrett's Esophagus on pathology. Fig. 6 graphs the mean CDX2 AACt in the two groups of patients segregated by the diagnosis of Barrett's Esophagus on pathology. Statistical analysis of the data showed the correlation between increased MUC2 and/or CDX2 expression and Barrett's esophagus confirmed by pathology was highly significant with a P value of < 0.001 by Student's 2 tailed t-test. In contrast, the expression of BMP4 does not differ significantly between patients with and without confirmed Barrett's esophagus.

[0080] The data was further analyzed by generation of Receiver Operating Characteristic (ROC) curves according to the operating characteristics set out in Table 4 and Table 5 for MUC2 and Table 6 and Table 7 for CDX2. ROC curves serve to plot the true positive rate against the false positive rate for different possible cutpoints of a diagnostic test. A ROC curve demonstrates the tradeoff between sensitivity and specificity (with increases in sensitivity typically accompanied by decreases in specificity). The closer the curve follows the left-hand border and then the top border of the ROC space, the more accurate the test. In contrast, the closer the curve comes to the 45-degree diagonal of the ROC space, the less accurate the test. The area under the curve (AUC) represents test accuracy.

[0081] Table 4: ROC curve Case Processing Summary for MUC2

Smaller values of the test result variable(s) indicate stronger evidence for a positive actual state.

a. The positive actual state is Yes.

[0082] Table 5: Area Under the Curve MUC2 AACt

Test accuracy of 92.8%

[0083] Table 6: ROC curve Case Processing Summary for CDX2

a. The positive actual state is Yes.

[0084] Table 7: Area Under the Curve CDX2 AACt

The test result variable(s): CDX2 Delta CT has at least one tie between the positive actual state group and the negative actual state group. Statistics may be biased.

a. Under the nonparametric assumption

b. Null hypothesis: true area = 0.5

Test accuracy of 88.1%

[0085] The analysis identified two very useful markers for Barrett's esophagus are MUC2 and CDX2. To highlight the remarkably positive results. The two tests are 100% sensitive with a 65% specificity making these very useful as screening tests for Barrett's esophagus. More importantly there were a very small number of subjects to get such strong results.

EXAMPLE 3

Further analysis of expanded cohort RT-qPCR testing for MUC2, CDX2 and BMP4.

[0086] Data from the cohort of patients presented in Example 2 were analyzed comparing expression of MUC2, CDX2 and BMP4 in esophageal brushings from suspected BE tissue proven by histology versus expression of these markers in brushing samples for normal squamous mucosa. When the BE esophageal brushing data is compared with the AACt of esophageal brushings of normal squamous epithelium, the data provided that the AACt cutpoint for MUC-2 and CDX-2 is 10 or less and is very striking graphically with only one outlier patient (the same patient for both MUC-2 and CDX-2). The AACt is a value obtained for both the BE esophageal brushings and normal squamous epithelium by subtracting out the house keeping gene values. The ACt may be otherwise termed the normalized delta Ct or alternatively as AACt. As used herein, the term "ACt cutpoint of 10 or less" is defined as that obtained when the BE esophageal brushing data is compared with the ACt of esophageal brushings of normal squamous epithelium.

[0087] Fig. 9A shows the RT-qPCR results for detection of MUC2 by brushings obtained from 17 patients with histologically confirmed BE compared to brushings from 23 patients with normal squamous epithelium. Fig. 9B shows the ACt for MUC2 expression in BE brushings compared to normal squamous brushings. Table 8 shows the data presented in Fig. 9 A and

Fig. 9B for normal squamous epithelium.

Table 8: ACt (AVG Target Gene - AVG ActB Ref.) MUC2 Normal Squamous Epithelium

Treatment AVG MUC2 Ct AVG ACTB Ct Δ Ct (AVG Target Gene - AVG ActB Reference)

BE17 Squamous 29.48 14.94 14.54

BE19 Squamous 28.67 11.76 16.90

BE21 Squamous 29.59 13.09 16.50

BE22 Squamous 40.00 22.80 17.20

BE26 Squamous 40.00 15.07 24.93

BE31 Squamous 32.80 13.23 19.57

BE34 Squamous 28.92 12.00 16.92

BE35 Squamous 40.00 17.32 22.68

BE36 Squamous 29.07 13.26 15.81

BE37 Squamous 26.66 13.71 12.95

BE38 Squamous 28.44 13.98 14.46

N = 23 Ave =17.28

[0088] Table 9 shows the data presented in Fig. 9 A and Fig. 9B for BE epithelium.

[0089] For the data presented in Tables 8 and 9, the lesion target gene Δ change is -14.09 with the fold increase in expression of MUC2 in BE tissue (2^Λ - AACt) is 18632.05. The significance of the difference by Students Paired t-test is <0.0001 (7.87 x 10^"17).

[0090] Fig. 10A shows the RT-qPCR results for detection of CDX2 by brushings obtained from 17 patients with histologically confirmed BE compared to brushings from 23 patients with normal squamous epithelium. Fig. 10B shows the ACt for CDX2 expression in BE brushings compared to normal squamous brushings. Table 10 shows the data presented in Fig. 10A and Fig. 10B for normal squamous epithelium.

Table 10: ACt (AVG Target Gene - AVG ActB Ref.) CDX2 NL Squamous Epithelium

[0091] Table 11 shows the data presented in Fig. 10A and Fig. 10B for BE epithelium.

Table 11: ACt (AVG Target Gene - AVG ActB Ref.) CDX2 BE Epithelium

Treatment AVG CDX2 AVG ACTB Δ Ct (AVG Target Gene - AVG

Ct Ct ActB Reference)

BE27 Lesion 19.99 14.47 5.52

BE28 Lesion 28.67 20.34 8.33

BE29 Lesion 23.62 15.87 7.75

BE30 Lesion 18.60 13.10 5.50

BE32 Lesion 24.53 16.48 8.06

BE33 Lesion 21.26 14.60 6.66

BE39 Lesion 16.77 12.67 4.10

BE40 Lesion 17.50 13.79 3.71

N = 17 Ave = 6.30

[0092] For the data presented in Tables 10 and 11, the lesion target gene Δ change is -15.02 with the fold increase in expression of CDX2 in BE tissue (2^A - AACt) is 33266.4836. The significance of the difference by students t-test is <0.0001 (7.39 x 10 -15).

[0093] Fig. 11A shows the RT-qPCR results for detection of BMP4 by brushings obtained from 17 patients with histologically confirmed BE compared to brushings from 23 patients with normal squamous epithelium. Fig. 11B shows the ACt for BMP4 expression in BE brushings compared to normal squamous brushings. Table 12 shows the data presented in Fig. 11A and Fig. 11B for normal squamous epithelium.

Table 12: ACt (AVG Target Gene - AVG ActB Ref.) BMP4 NL Squamous Epithelium

BE34 Squamous 28.00 12.00 16.00

BE35 Squamous 32.65 17.32 15.33

BE36 Squamous 26.16 13.26 12.90

BE37 Squamous 36.27 13.71 22.55

BE38 Squamous 31.03 13.98 17.05

N = 23 Ave = 16.59

[0094] Table 13 shows the data presented in Fig. 11A and Fig. 11B for BE epithelium.

[0095] For the data presented in Tables 12 and 12, the lesion target gene Δ chang with the fold increase in expression of BMP4 in BE tissue (2^A - AACt) is 111.143. The significance of the difference by students t-test is <0.0001 (5.33 x 10^"8).

[0096] Recitation of the claims:

Claim 1. A method for diagnosing Barrett's Esophagus (BE) comprising: testing a sample of cells from an esophagus of a patient suspected of having BE for expression of an intestinal marker in the esophagus; comparing a relative expression of the intestinal marker with that of a constitutively expressed marker; establishing a ratio of the relative expression; and diagnosing the patient as having as having BE if the ratio of relative expression shows a presence of intestinal metaplasia in the esophagus. [0097] Claim 2. The method of claim 1, wherein the sample is collected by esophageal brushing.

[0098] Claim 3. The method of claim 1, wherein the sample is collected by retrieval of a tethered cell collection device that is first swallowed, allowed to expand and then retrieved.

[0099] Claim 4. The method of claim 3, wherein the tethered cell collection device is a sponge compacted into a gelatin capsule that dissolves after being swallowed.

[00100] Claim 5. The method of claim 1, wherein the intestinal marker is one or more of the group consisting of MUC2 and CDX2.

[00101] Claim 6. The method of claim 1, wherein the intestinal marker is two or more of the group consisting of MUC2, CDX2 and BMP4.

[00102] Claim 7. The method of any one of claims 1 - 6, wherein the testing for expression of an intestinal marker in the esophagus is conducted by quantitative reverse transcriptase PCR (RT-qPCR).

[00103] Claim 8. The method of claim 7, wherein the patient is diagnosed as having BE when the ratio of relative expression is at ACt cutpoint of less than 10.

[00104] Claim 9. The method in any one of claims 1 to 8, wherein the protein is MUC2.

[00105] Claim 10. The method in any one of claims 1 to 8, wherein the protein is CDX2.

[00106] Claim 11. A method for diagnosing the presence of Barrett's Esophagus (BE) in a subject at risk of having BE, the method comprising: a) contacting a sample derived from esophageal cells collected from the subject with at least one biomarker reagent, wherein the at least one biomarker reagent includes a reagent that specifically binds to a MUC2 biomarker in the sample; b) measuring the level of the MUC2 biomarker in the sample; and c) detecting that the level of the MUC2 biomarker is increased in the esophageal sample relative to a control sample, thereby detecting the presence of BE in the subject.

[00107] Claim 12. The method of claim 11, wherein the sample is collected by esophageal brushing.

[00108] Claim 13. The method of claim 11, wherein the at least one biomarker reagent further includes a reagent that specifically binds to a CDX2 biomarker in the sample.

[00109] Claim 14. The method of claim 11, wherein the at least one biomarker reagent further includes a reagent that specifically binds to a BMP4 biomarker in the sample. [00110] Claim 15. The method of any one of claims 11 - 14, wherein the biomarker level is measured by RT-qPCR.

[00111] Claim 16. The method of any one of claims 11 - 14, wherein the biomarker level is measured by immunoassay, including by ELISA.

[00112] Claim 17. The method of claim 11, wherein the sample derived from esophageal cells collected from the subject is contacted with at least two biomarker reagents, wherein the at least two biomarker reagents include reagents that specifically bind to a MUC2 biomarker and a CDX2 biomarker and the MUC2 and CDX2 biomarkers are measured by RT-qPCR.

[00113] Claim 18. The method of claim 11, wherein the sample derived from esophageal cells collected from the subject is contacted with at least one additional biomarker reagent, wherein the at least one additional biomarker reagent is selected from a CDX2 biomarker reagent and a BMP4 biomarker reagent.

[00114] Claim 19. A method for diagnosing the presence of Barrett's Esophagus (BE) in a subject at risk of having BE, the method comprising: a) contacting a sample derived from esophageal cells collected from the subject with at least one biomarker reagent, wherein the at least one biomarker reagent includes a reagent that specifically binds to a MUC2 biomarker in the sample; b) measuring the level of the MUC2 biomarker in the sample by RT-qPCR; and c) detecting that the level of the MUC2 biomarker is increased in the esophageal sample relative to a control sample, thereby detecting the presence of BE in the subject.

[00115] Claim 20. A method for diagnosing the presence of Barrett's Esophagus (BE) in a subject at risk of having BE, the method comprising: a) contacting a sample derived from esophageal cells collected from the subject with at least one biomarker reagent, wherein the at least one biomarker reagent includes a reagent that specifically binds to a MUC2 biomarker in the sample; b) measuring the level of the MUC2 biomarker in the sample by immunoassay; and c) detecting that the level of the MUC2 biomarker is increased in the esophageal sample relative to a control sample, thereby detecting the presence of BE in the subject.

[00116] All publications, patents and patent applications cited herein are hereby incorporated by reference as if set forth in their entirety herein. While this invention has been described with reference to illustrative embodiments, this description is not intended to be construed in a limiting sense. Various modifications and combinations of illustrative embodiments, as well as other embodiments of the invention, will be apparent to persons skilled in the art upon reference to the description.

Claims

Claims:

1. A method for diagnosing Barrett's Esophagus (BE) comprising:

testing a sample of cells from an esophagus of a patient suspected of having BE for expression of an intestinal marker in the esophagus;

comparing a relative expression of the intestinal marker with that of a constitutively expressed marker;

establishing a ratio of the relative expression; and

diagnosing the patient as having as having BE if the ratio of relative expression shows a presence of intestinal metaplasia in the esophagus.

2. The method of claim 1, wherein the sample is collected by esophageal brushing.

3. The method of claim 1, wherein the sample is collected by retrieval of a tethered cell collection device that is first swallowed, allowed to expand and then retrieved.

4. The method of claim 3, wherein the tethered cell collection device is a sponge compacted into a gelatin capsule that dissolves after being swallowed.

5. The method of claim 1, wherein the intestinal marker is one or more of the group consisting of MUC2 and CDX2.

6. The method of claim 1, wherein the intestinal marker is two or more of the group consisting of MUC2, CDX2 and BMP4.

7. The method of any one of claims 1 - 6, wherein the testing for expression of an intestinal marker in the esophagus is conducted by quantitative reverse transcriptase PCR (RT-qPCR).

8. The method of claim 7, wherein the patient is diagnosed as having BE when the ratio of relative expression is at ACt cutpoint of less than 10.

9. The method in any one of claims 1 to 8, wherein the protein is MUC2.

10. The method in any one of claims 1 to 8, wherein the protein is CDX2.

11. A method for diagnosing the presence of Barrett's Esophagus (BE) in a subject at risk of having BE, the method comprising: a) contacting a sample derived from esophageal cells collected from the subject with at least one biomarker reagent, wherein the at least one biomarker reagent includes a reagent that specifically binds to a MUC2 biomarker in the sample; b) measuring the level of the MUC2 biomarker in the sample; and c) detecting that the level of the MUC2 biomarker is increased in the esophageal sample relative to a control sample, thereby detecting the presence of BE in the subject.

12. The method of claim 11, wherein the sample is collected by esophageal brushing.

13. The method of claim 11, wherein the at least one biomarker reagent further includes a reagent that specifically binds to a CDX2 biomarker in the sample.

14. The method of claim 11, wherein the at least one biomarker reagent further includes a reagent that specifically binds to a BMP4 biomarker in the sample.

15. The method of any one of claims 11 - 14, wherein the biomarker level is measured by RT-qPCR.

16. The method of any one of claims 11 - 14, wherein the biomarker level is measured by immunoassay.

17. The method of claim 11, wherein the sample derived from esophageal cells collected from the subject is contacted with at least two biomarker reagents, wherein the at least two biomarker reagents include reagents that specifically bind to a MUC2 biomarker and a CDX2 biomarker and the MUC2 and CDX2 biomarkers are measured by RT-qPCR.

18. The method of claim 11, wherein the sample derived from esophageal cells collected from the subject is contacted with at least one additional biomarker reagent, wherein the at least one additional biomarker reagent is selected from a CDX2 biomarker reagent and a BMP4 biomarker reagent.

19. A method for diagnosing the presence of Barrett's Esophagus (BE) in a subject at risk of having BE, the method comprising: a) contacting a sample derived from esophageal cells collected from the subject with at least one biomarker reagent, wherein the at least one biomarker reagent includes a reagent that specifically binds to a MUC2 biomarker in the sample; b) measuring the level of the MUC2 biomarker in the sample by RT-qPCR; and c) detecting that the level of the MUC2 biomarker is increased in the esophageal sample relative to a control sample, thereby detecting the presence of BE in the subject.

20. A method for diagnosing the presence of Barrett's Esophagus (BE) in a subject at risk of having BE, the method comprising: a) contacting a sample derived from esophageal cells collected from the subject with at least one biomarker reagent, wherein the at least one biomarker reagent includes a reagent that specifically binds to a MUC2 biomarker in the sample; b) measuring the level of the MUC2 biomarker in the sample by immunoassay; and c) detecting that the level of the MUC2 biomarker is increased in the esophageal sample relative to a control sample, thereby detecting the presence of BE in the subject.