WO2016183487A1 - Compositions and methods for detecting sessile serrated adenomas/polyps - Google Patents

Abstract

The disclosure provides a method to detect sessile serrated adenomas/polyps (SSA/Ps) and to differentiate SSA/Ps from hyperplastic polyps (HPs). The method uses the differential staining patterns of MUC5AC and TFF1.

Description

COMPOSITIONS AND METHODS FOR DETECTING SESSILE SERRATED

ADENOMAS/POLYPS

GOVERNMENTAL RIGHTS

[0001 ] This invention was made with government support under

R21 CA176130 awarded by the NIH. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application number 62/161 ,016, filed May 13, 2015, the disclosure of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The disclosure provides a method to detect sessile serrated adenomas/polyps (SSA/Ps) and to differentiate SSA/Ps from hyperplastic polyps (HPs). The method uses the differential staining patterns of MUC5AC and TFF1 .

BACKGROUND OF THE INVENTION

[0004] Colon cancer is the second largest cause of cancer-related deaths in the United States. Colonic neoplasms originate primarily from colon polyps, and develop via partially overlapping but mechanistically distinct pathways that have been designated as the adenomatous and serrated pathways. Accumulating evidence indicates that the majority of other colon adenocarcinomas, possibly 20-30%, arise from a subset of serrated polyps, designated sessile serrated adenomas/polyps (SSA/Ps), which were previously classified as hyperplastic polyps and thought to have little or no tumorigenic potential.

[0005] Differentiating between SSA/Ps and HPs on routine histological examination can be challenging, particularly in small or fragmented samples. This has been highlighted by a number of studies documenting the frequent misclassification of SSA/Ps as HPs, resulting in inadequate follow-up. Conversely, misclassifying an HP as an SSA/P may result in unnecessary cancer screening in these patients.

l [0006] Thus, there is a need in the art for reliable diagnostic assays that could aid in the distinction between these lesions. Such an assay would be helpful for both diagnosis and surveillance stratification of patients.

SUMMARY OF THE INVENTION

[0007] In an aspect, the disclosure provides a method of detecting sessile serrated adenomas/polyps (SSA/Ps) in a subject. The method comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; and (b) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is greater than 20% relative to baseline and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp.

[0008] In another aspect, the disclosure provides a method of

differentiating sessile serrated adenomas/polyps (SSA/Ps) from hyperplastic polyps (HPs) in a subject. The method comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; and (b) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is greater than 20% relative to baseline and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp.

[0009] In still another aspect, the disclosure provides a method of predicting the likelihood that a colorectal polyp in a subject will develop into colorectal cancer. The method comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; and (b) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is greater than 20% relative to baseline and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp, wherein the detection of SSA/Ps in the subject indicates an increased likelihood of developing colorectal cancer.

[0010] In a different aspect, the disclosure provides a method of detecting sessile serrated adenomas/polyps (SSA/Ps) in a subject. The method comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; (b) comparing the amount of MUC5AC and TFF1 protein in the biological sample to a reference value; and (c) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is significantly increased relative to the reference value and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp.

[001 1 ] In another different aspect, the disclosure provides a method of differentiating sessile serrated adenomas/polyps (SSA/Ps) from hyperplastic polyps (HPs) in a subject. The method comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; (b) comparing the amount of MUC5AC and TFF1 protein in the biological sample to a reference value; (c) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is significantly increased relative to the reference value and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp.

[0012] In still another different aspect, the disclosure provides a method of predicting the likelihood that a colorectal polyp in a subject will develop into colorectal cancer. The method comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; (b) comparing the amount of MUC5AC and TFF1 protein in the biological sample to a reference value; and (c) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is significantly increased relative to the reference value and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp, wherein the detection of SSA/Ps in the subject indicates an increased likelihood of developing colorectal cancer.

BRIEF DESCRIPTION OF THE FIGURES

[0013] The application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] FIG. 1A, FIG. 1 B and FIG. 1C depict expression of MUC5AC and

TFF1 in normal colon (FIG. 1A), HPs (FIG. 1 B) and SSA/Ps (FIG. 1C). The immunofluorescence and immunoperoxidase signals for the gastric proteins MUC5AC and TFF1 were negligible in normal colonic mucosa. However, their expression was evident in serrated polyps. On average, cells that were immunopositive in HP samples were scattered and often located at the luminal surface of polyps. In SSA/P samples, MUC5AC and TFF1 were typically expressed by most of the epithelial cells along the entire length of architecturally compromised crypts. SSA/Ps, compared to HPs, also showed significant coexpression of both MUC5AC and TFF1 in merged

immunofluorescence analyses.

[0015] FIG. 2 depicts bar graphs illustrating the differences in signal intensities for MUC5AC and TFF1 in normal colon, HPs and SSA/Ps. The images were scored by experienced Gl pathologists based on the percentage of serrated crypt cells with immunostaining (0: none; 1 : 1 - 25%; 2: 26-50%; 3: 51 -75%; 4: >76%) and intensity of staining (0-4) (^*) p (two-tailed) < 0.02.

[0016] FIG. 3A and FIG. 3B depict representative examples of co- localization of MUC5AC and TFF1 in HPs (FIG. 3A) and SSA/Ps (FIG. 3B). Note that compared to HPs, majority of SSA/P samples showed significant co-expression often approaching 100%. HPs also exhibited greater variability in distribution and co- localization of signals. In most cases, expression of MUC5AC and TFF1 was observed in separate cells with little co-localization. Two of 46 HPs analyzed demonstrated more co-expression of MUC5AC and TFF1 suggestive of but not identical to SSA/Ps (FIG. 3A, 2^nd and 3^rd row).

DETAILED DESCRIPTION OF THE INVENTION

[0017] Provided herein are methods to detect sessile serrated

adenomas/polyps (SSA/Ps) and to distinguish SSA/Ps from hyperplastic polyps (HPs). Prior to the disclosure, there has been difficulty in distinguishing SSA/Ps from HPs. Current methods have about a 60-70% accuracy in distinguishing SSA/Ps from HPs. However, the methodology disclosed herein has a greater than 90% accuracy at correctly distinguishing SSA/Ps from HPs. As SSA/Ps have a higher risk of progressing to cancer, it is important that SSA/Ps are accurately diagnosed such that the subject is treated properly. By accurately detecting SSA/Ps, the subject may be treated more aggressively or monitored more frequently. Thus, the method disclosed herein may be used to determine the risk of progression to colorectal cancer and also decrease the risk of progression to colorectal cancer by allowing for earlier interventions.

[0018] Details of the methods are described in more detail below.

I. METHODS

[0019] In an aspect, the disclosure provides a method to classify a subject based on the amount and pattern of MUC5AC and TFF1 protein measured in a biological sample obtained from the subject. The method generally comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in the biological sample, and (b) classifying the subject as having (i) an increased or decreased amount of MUC5AC and TFF1 protein based on the amount of MUC5AC and TFF1 protein measured in the sample and/or (ii) co-localized or non-co-localized MUC5AC and TFF1 protein and/or MUC5AC and TFF1 protein throughout the polyp or only on the luminal surface of the polyp.

[0020] In another aspect, the disclosure provides a method of detecting sessile serrated adenomas/polyps (SSA/Ps) in a subject. The method comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; and (b) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is greater than 20% relative to baseline and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp.

[0021 ] In still another aspect, the disclosure provides a method of differentiating sessile serrated adenomas/polyps (SSA/Ps) from hyperplastic polyps (HPs) in a subject. The method comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; and (b) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is greater than 20% relative to baseline and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp.

[0022] In still yet another aspect, the disclosure provides a method of predicting the likelihood that a colorectal polyp in a subject will develop into colorectal cancer. The method comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; and (b) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is greater than 20% relative to baseline and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp, wherein the detection of SSA/Ps in the subject indicates an increased likelihood of developing colorectal cancer. Treatment decisions may then be made based on the detection of SSA/Ps.

[0023] In a different aspect, the disclosure provides a method to classify a subject based on the amount and pattern of MUC5AC and TFF1 protein measured in a biological sample obtained from the subject. The method generally comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in the biological sample, (b) comparing the amount of MUC5AC and TFF1 protein in the biological sample to a reference value, and (c) classifying the subject as having (i) an increased or decreased amount of MUC5AC and TFF1 protein based on the amount of MUC5AC and TFF1 protein measured in the sample and/or (ii) co-localized or non-co-localized MUC5AC and TFF1 protein and/or MUC5AC and TFF1 protein throughout the polyp or only on the luminal surface of the polyp.

[0024] In another different aspect, the disclosure provides a method of detecting sessile serrated adenomas/polyps (SSA/Ps) in a subject. The method generally comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; (b) comparing the amount of MUC5AC and TFF1 protein in the biological sample to a reference value; and (c) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is significantly increased relative to the reference value and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp.

[0025] In still another different aspect, the disclosure provides a method of differentiating sessile serrated adenomas/polyps (SSA/Ps) from hyperplastic polyps (HPs) in a subject. The method generally comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; (b) comparing the amount of MUC5AC and TFF1 protein in the biological sample to a reference value; and (c) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is significantly increased relative to the reference value and/or the expression pattern of MUC5AC and TFF1 protein is co- localized and detectable throughout the polyp.

[0026] In still yet another different aspect, the disclosure provides a method of predicting the likelihood that a colorectal polyp in a subject will develop into colorectal cancer. The method generally comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; (b) comparing the amount of MUC5AC and TFF1 protein in the biological sample to a reference value; and (c) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is significantly increased relative to the reference value and/or the expression pattern of MUC5AC and TFF1 protein is co- localized and detectable throughout the polyp, wherein the detection of SSA/Ps in the subject indicates an increased likelihood of developing colorectal cancer. Treatment decisions may then be made based on the detection of SSA/Ps.

[0027] In other aspects, the disclosure provides a method of determining treatment of a subject diagnosed with serrated polyps or suspected of having serrated polyps. The method generally comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; and (b) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is greater than 20% relative to baseline and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp; and (c) treating the subject more aggressively if SSA/Ps are detected. Alternatively, the disclosure provides a method of determining treatment of a subject diagnosed with serrated polyps or suspected of having serrated polyps. The method generally comprises: (a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; (b) comparing the amount of MUC5AC and TFF1 protein in the biological sample to a reference value; and (c) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is significantly increased relative to the reference value and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp; and (d) treating the subject more aggressively if SSA/Ps are detected. Serrated polyps may be classified into hyperplastic polyps (HPs), sessile serrated adenomas/polyps (SSA/Ps), and traditional serrated adenomas (TSAs). SSA/Ps have the strongest association with an increased risk for colon cancer. Accordingly, if SSA/Ps are detected, the subject may be more aggressively treated relative to treatment for HPs. Non-limiting examples of treatment for SSA/Ps include polypectomy, endoscopic resection, and surgical resection, all followed with surveillance. Additionally, if SSA/Ps are detected, the subject may be subjected to an increased frequency of surveillance, such as colonoscopy. For example, the subject may receive a colonoscopy every about 1 to about 6 years.

Accordingly, if SSA/Ps are detected, the subject may receive a colonoscopy about every 1 year, about every 2 years, about every 3 years, about every 4 years, about every 5 years, or about every 6 years. For example, a subject having a polyp classified as an SSA/P according to the methods detailed herein and the polyp having diameter of at least about 10 mm would have a subsequent colonoscopy in about 2 years to about 4 years, or about 3 years. For example, a subject having a polyp classified as an SSA/P according to the methods detailed herein and the polyp having of diameter of less than about 5 mm would have a subsequent colonoscopy in about 4 years to about 6 years, or about 5 years. A subject having a polyp classified as an SSA/P according to the methods detailed herein and being of diameter of about 5 mm to about 10 mm would have a subsequent colonoscopy in about 2 years to about 6 years, about 3 to about 5 years, or about 4 years. More frequent colonoscopies may be suggested for subjects having multiple SSA/P polyps. By more accurately diagnosing a polyp as a SSA/P instead of as a hyperplastic polyp, a subject may be more frequently screened by colonoscopy, leading to a reduced incidence of colon cancer and deaths due to colon cancer.

[0028] In other aspects, the disclosure provides a method for monitoring serrated polyps in a subject. In such an embodiment, a method of detecting sessile serrated adenomas/polyps (SSA/Ps) in a subject is performed at one point in time. Then, at a later time, the method of detecting sessile serrated adenomas/polyps

(SSA/Ps) in the subject may be performed to determine the change in serrated polyps over time. For example, the method of detecting sessile serrated adenomas/polyps (SSA/Ps) may be performed on the same subject days, weeks, months, or years following the initial use of the method to detect sessile serrated adenomas/polyps (SSA/Ps). Accordingly, the method of detecting SSA/Ps may be used to follow a subject over time to determine when the risk of progressing to more severe disease is high thereby requiring treatment. Additionally, the method of detecting SSA/Ps may be used to measure the rate of disease progression. For example, an increased amount of MUC5AC and TFF1 protein and co-localized MUC5AC and TFF1 protein and/or MUC5AC and TFF1 protein throughout the polyp may indicate disease progression. Early assessment of the risk of colorectal cancer in the subject may reduce the development and/or progression of symptoms associated with colorectal cancer by enabling improved interventions or enabling earlier interventions.

[0029] Additionally, a method for monitoring serrated polyps in a subject may be used to determine the response to treatment. As used herein, subjects who respond to treatment are said to have benefited from treatment. For example, a method of detecting SSA/Ps may be performed on the biological sample of the subject prior to initiation of treatment. Then, at a later time, a method of detecting SSA/Ps may be used to determine the response to treatment over time. For example, a method of detecting SSA/Ps may be performed on the biological sample of the same subject days, weeks, months, or years following initiation of treatment. Accordingly, a method of detecting SSA/Ps may be used to follow a subject receiving treatment to determine if the subject is responding to treatment. If the amount of MUC5AC and TFF1 protein and co- localization of MUC5AC and TFF1 protein and/or MUC5AC and TFF1 protein

throughout the polyp increases or remains the same, then the subject may not be responding to treatment. If the amount of MUC5AC and TFF1 protein and co- localization of MUC5AC and TFF1 protein and/or MUC5AC and TFF1 protein

throughout the polyp decreases, then the subject may be responding to treatment.

These steps may be repeated to determine the response to therapy over time. [0030] In any of the foregoing embodiments, the subject may or may not be diagnosed with serrated polyps or SSA/Ps. In certain embodiments, the subject may not be diagnosed with serrated polyps or SSA/Ps but is suspected of having serrated polyps or SSA/Ps based on symptoms. Non-limiting examples of symptoms of serrated polyps or SSA/Ps that may lead to a diagnosis include bleeding and iron deficiency anemia. In other embodiments, the subject may not be diagnosed with serrated polyps or SSA/Ps but is at risk of having serrated polyps or SSA/Ps. Non-limiting examples of risk factors for serrated polyps or SSA/Ps include smoking, diabetes, obesity, age, sex, diet, and family history. In other embodiment, the subject has no symptoms and/or no risk factors for serrated polyps or SSA/Ps. Methods of diagnosing serrated polyps or SSA/Ps are known in the art. Non-limiting examples of methods of diagnosing serrated polyps or SSA/Ps include histological pathology.

[0031 ] Suitable subjects include, but are not limited to, a human, a livestock animal, a companion animal, a lab animal, and a zoological animal. In one embodiment, the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas. In yet another embodiment, the subject may be a companion animal. Non- limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoological animal. As used herein, a "zoological animal" refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In an embodiment, the animal is a laboratory animal. Non-limiting examples of a laboratory animal may include rodents, canines, felines, and non-human primates. In certain embodiments, the animal is a rodent. In a preferred embodiment, the subject is human.

(a) biological sample

[0032] As used herein, the term "biological sample" refers to a sample obtained from a subject. Any biological sample containing tissue from a colorectal polyp is suitable. Numerous types of biological samples are known in the art. Suitable biological sample may include, but are not limited to, tissue samples. In some embodiments, the biological sample is a tissue sample such as a tissue biopsy. The biopsied tissue may be fixed, embedded in paraffin or plastic, and sectioned, or the biopsied tissue may be frozen and cryosectioned. In a specific embodiment, the biopsied tissue is from a colorectal polyp.

[0033] As will be appreciated by a skilled artisan, the method of collecting a biological sample can and will vary depending upon the nature of the biological sample and the type of analysis to be performed. Any of a variety of methods generally known in the art may be utilized to collect a biological sample. Generally speaking, the method preferably maintains the integrity of the sample such that the MUC5AC and TFF1 protein can be accurately detected and the amount measured according to the disclosure.

[0034] In some embodiments, a single sample is obtained from a subject to detect MUC5AC and TFF1 protein in the sample. Alternatively, MUC5AC and TFF1 protein may be detected in samples obtained over time from a subject. As such, more than one sample may be collected from a subject over time. For instance, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16 or more samples may be collected from a subject over time. In some embodiments, 2, 3, 4, 5, or 6 samples are collected from a subject over time. In other embodiments, 6, 7, 8, 9, or 10 samples are collected from a subject over time. In yet other embodiments, 10, 1 1 , 12, 13, or 14 samples are collected from a subject over time. In other embodiments, 14, 15, 16 or more samples are collected from a subject over time.

[0035] When more than one sample is collected from a subject over time, samples may be collected every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or more days. In some embodiments, samples are collected every 1 , 2, 3, 4, or 5 days. In other embodiments, samples are collected every 5, 6, 7, 8, or 9 days. In yet other

embodiments, samples are collected every 9, 10, 1 1 , 12 or more days. In still other embodiments, samples are collected a month apart, 3 months apart, 6 months apart, 1 year apart, 2 years apart, 5 years apart, 10 years apart or more. (b) detecting MUC5AC and TFF1 protein

[0036] Once a sample is obtained, it is processed in vitro to detect and measure the amount of MUC5AC and TFF1 protein. All suitable methods for detecting and measuring an amount of MUC5AC and TFF1 protein known to one of skill in the art are contemplated within the scope of the invention. Non-limiting examples of suitable methods to assess an amount of protein expression may include epitope binding agent- based methods. As used herein the "amount" of MUC5AC and TFF1 protein includes the number of cells expressing MUC5AC and TFF1 protein and/or the intensity or level of MUC5AC and TFF1 protein staining or expression in the biological sample.

[0037] In some embodiments, the method to assess an amount of protein expression is an epitope binding agent-based method. As used herein, the term

"epitope binding agent" refers to an antibody, an aptamer, a nucleic acid, an

oligonucleic acid, an amino acid, a peptide, a polypeptide, a protein, a lipid, a

metabolite, a small molecule, or a fragment thereof that recognizes and is capable of binding to a target gene protein. Nucleic acids may include RNA, DNA, and naturally occurring or synthetically created derivative.

[0038] As used herein, the term "antibody" generally means a polypeptide or protein that recognizes and can bind to an epitope of an antigen. An antibody, as used herein, may be a complete antibody as understood in the art, i.e., consisting of two heavy chains and two light chains, or may be any antibody-like molecule that has an antigen binding region, and includes, but is not limited to, antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies, Fv, and single chain Fv. The term antibody also refers to a polyclonal antibody, a monoclonal antibody, a chimeric antibody and a humanized antibody. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and

characterizing antibodies are also well known in the art (See, e.g. Antibodies: A

Laboratory Manual, Cold Spring Harbor Laboratory, 1988; herein incorporated by reference in its entirety).

[0039] As used herein, the term "aptamer" refers to a polynucleotide, generally a RNA or DNA that has a useful biological activity in terms of biochemical activity, molecular recognition or binding attributes. Usually, an aptamer has a molecular activity such as binging to a target molecule at a specific epitope (region). It is generally accepted that an aptamer, which is specific in it binding to a polypeptide, may be synthesized and/or identified by in vitro evolution methods. Means for preparing and characterizing aptamers, including by in vitro evolution methods, are well known in the art (See, e.g. US 7,939,313; herein incorporated by reference in its entirety).

[0040] In general, an epitope binding agent-based method of assessing an amount of protein expression comprises contacting a sample comprising a polypeptide with an epitope binding agent specific for the polypeptide under conditions effective to allow for formation of a complex between the epitope binding agent and the polypeptide.

[0041 ] Contacting the sample with an epitope binding agent under effective conditions for a period of time sufficient to allow formation of a complex generally involves adding the epitope binding agent composition to the sample and incubating the mixture for a period of time long enough for the epitope binding agent to bind to any antigen present. After this time, the complex will be washed and the complex may be detected by any method well known in the art. Methods of detecting the epitope binding agent-polypeptide complex are generally based on the detection of a label or marker. The term "label", as used herein, refers to any substance attached to an epitope binding agent, or other substrate material, in which the substance is detectable by a detection method. Non-limiting examples of suitable labels include luminescent molecules, chemiluminescent molecules, fluorochromes, fluorescent quenching agents, colored molecules, radioisotopes, scintillants, biotin, avidin, stretpavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni2+, Flag tags, myc tags, heavy metals, and enzymes (including alkaline phosphatase, peroxidase, and luciferase). In one embodiment, a MUC5AC and TFF1 epitope binding agent comprise a peroxidase enzyme. In other embodiments, a MUC5AC epitope binding agent comprises a fluorophore that emits light at one wavelength and a TFF1 epitope binding agent comprises a fluorophore that emits light at a second wavelength, such that co-localization of MUC5AC and TFF1 may be observed. For example, a MUC5AC epitope binding agent comprises a fluorophore that emits green light and a TFF1 epitope binding agent comprises a fluorophore that emits red light, such that co- localization of MUC5AC and TFF1 results in the observation of yellow light.

Alternatively, a MUC5AC epitope binding agent comprises a fluorophore that emits red light and a TFF1 epitope binding agent comprises a fluorophore that emits green light, such that co-localization of MUC5AC and TFF1 results in the observation of yellow light. Other fluorophore combinations are known to those of skill in the art.

[0042] Alternatively, the MUC5AC and TFF1 epitope binding agents do not comprise a label, but instead are contacted with a secondary epitope binding agent that specifically recognizes the MUC5AC and TFF1 epitope binding agents and comprises a label. For example, a MUC5AC antibody is contacted with a secondary antibody conjugated to a fluorophore that specifically recognizes the constant region of the MUC5AC antibody. In the same manner, a TFF1 antibody is contacted with a secondary antibody conjugated to a fluorophore that specifically recognizes the constant region of the TFF1 antibody. Alternatively, a MUC5AC antibody is contacted with a secondary antibody conjugated to a peroxidase enzyme that specifically recognizes the constant region of the MUC5AC antibody. In the same manner, a TFF1 antibody is contacted with a secondary antibody conjugated to a peroxidase enzyme that specifically recognizes the constant region of the TFF1 antibody.

[0043] Methods of detecting an epitope binding agent-polypeptide complex based on the detection of a label or marker are well known in the art.

[0044] In a specific embodiment, the epitope binding agent-based method is immunohistochemistry (IHC). IHC uses an antibody to detect and quantify antigens in intact tissue samples. The tissue samples may be fresh-frozen and/or formalin-fixed, paraffin-embedded (or plastic-embedded) tissue blocks prepared for study by IHC.

Methods of preparing tissue block for study by IHC, as well as methods of performing IHC are well known in the art.

[0045] Additionally, MUC5AC and TFF1 nucleic acid expression may be measured to determine the amount of MUC5AC and TFF1 protein in a biological sample. In a specific embodiment, MUC5AC and TFF1 mRNA may be measured to determine the amount of MUC5AC and TFF1 protein in a biological sample. [0046] Methods for assessing an amount of nucleic acid expression in a sample are well known in the art, and all suitable methods for assessing an amount of nucleic acid expression known to one of skill in the art are contemplated within the scope of the invention. The term "amount of nucleic acid expression" or "level of nucleic acid expression" as used herein refers to a measurable level of expression of the nucleic acids, such as, without limitation, the level of messenger RNA (mRNA) transcript expressed or a specific variant or other portion of the mRNA, the enzymatic or other activities of the nucleic acids, and the level of a specific metabolite. The term "nucleic acid" includes DNA and RNA and can be either double stranded or single stranded. Non-limiting examples of suitable methods to assess an amount of nucleic acid expression may include arrays, such as microarrays, PCR, such as RT-PCR (including quantitative RT-PCR), nuclease protection assays and Northern blot analyses. In a specific embodiment, determining the amount of expression of a target nucleic acid comprises, in part, measuring the level of target nucleic acid mRNA expression.

[0047] In one embodiment, the amount of nucleic acid expression may be determined by using an array, such as a microarray. Methods of using a nucleic acid microarray are well and widely known in the art. For example, a nucleic acid probe that is complementary or hybridizable to an expression product of a target gene may be used in the array. The term "hybridize" or "hybridizable" refers to the sequence specific non-covalent binding interaction with a complementary nucleic acid. In a preferred embodiment, the hybridization is under high stringency conditions. Appropriate stringency conditions which promote hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. The term "probe" as used herein refers to a nucleic acid sequence that will hybridize to a nucleic acid target sequence. In one example, the probe hybridizes to an RNA product of the nucleic acid or a nucleic acid sequence

complementary thereof. The length of probe depends on the hybridization conditions and the sequences of the probe and nucleic acid target sequence. In one embodiment, the probe is at least 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500 or more nucleotides in length. [0048] In another embodiment, the amount of nucleic acid expression may be determined using PCR. Methods of PCR are well and widely known in the art, and may include quantitative PCR, semi-quantitative PCR, multiplex PCR, or any

combination thereof. Specifically, the amount of nucleic acid expression may be determined using quantitative RT-PCR. Methods of performing quantitative RT-PCR are common in the art. In such an embodiment, the primers used for quantitative RT-PCR may comprise a forward and reverse primer for a target gene. The term "primer" as used herein refers to a nucleic acid sequence, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand is induced (e.g. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon factors, including temperature, sequences of the primer and the methods used. A primer typically contains 15-25 or more nucleotides, although it can contain less or more. The factors involved in determining the appropriate length of primer are readily known to one of ordinary skill in the art.

[0049] The amount of nucleic acid expression may be measured by measuring an entire mRNA transcript for a nucleic acid sequence, or measuring a portion of the mRNA transcript for a nucleic acid sequence. For instance, if a nucleic acid array is utilized to measure the amount of mRNA expression, the array may comprise a probe for a portion of the mRNA of the nucleic acid sequence of interest, or the array may comprise a probe for the full mRNA of the nucleic acid sequence of interest. Similarly, in a PCR reaction, the primers may be designed to amplify the entire cDNA sequence of the nucleic acid sequence of interest, or a portion of the cDNA sequence. One of skill in the art will recognize that there is more than one set of primers that may be used to amplify either the entire cDNA or a portion of the cDNA for a nucleic acid sequence of interest. Methods of designing primers are known in the art. Methods of extracting RNA from a biological sample are known in the art. [0050] The level of expression may or may not be normalized to the level of a control nucleic acid. This allows comparisons between assays that are performed on different occasions.

(c) detecting SSA/Ps

[0051 ] The amount of MUC5AC and TFF1 protein measured in the biological sample may be used to detect SSA/Ps. As described above, the amount of MUC5AC and TFF1 protein may include the number of cells expressing MUC5AC and TFF1 in the biological sample and/or the intensity or level of MUC5AC and TFF1 protein expression in the biological sample. In certain embodiments, SSA/Ps are detected when the amount of MUC5AC and TFF1 protein is greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, or greater than 50% relative to baseline. When the number of cells expressing MUC5AC and TFF1 are measured, baseline is the number of cells in the biological sample. When the intensity or level of MUC5AC and TFF1 protein are measured, baseline is the

background staining of the biological sample. In other embodiments, SSA/Ps are detected when the amount of MUC5AC protein is greater than 20%, greater than 25%, greater than 30%, or greater than 35% and the amount of TFF1 protein is greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, or greater than 50% relative to baseline. Specifically, SSA/Ps are detected when the amount of MUC5AC protein is greater than 30% and the amount of TFF1 protein is greater than 45% relative to baseline.

[0052] In another embodiment, SSA/Ps are detected when the amount of MUC5AC protein, as measured by the number of cells expressing MUC5AC, is greater than 30%, greater than 35%, or greater than 40% relative to baseline and the amount of TFF1 protein, as measured by the number of cells expressing TFF1 , is greater than 45%, greater than 50%, or greater than 55% relative to baseline. Specifically, SSA/Ps are detected when the amount of MUC5AC protein, as measured by the number of cells expressing MUC5AC, is greater than 30% relative to baseline and the amount of TFF1 protein, as measured by the number of cells expressing TFF1 , is greater than 45% relative to baseline. [0053] In still another embodiment, SSA/Ps are detected when the amount of MUC5AC protein, as measured by the intensity or level of MUC5AC protein expression, is greater than 20%, greater than 25%, or greater than 30% relative to baseline and the amount of TFF1 protein, as measured by the intensity or level TFF1 protein expression, is greater than 25%, greater than 30%, or greater than 35% relative to baseline. Specifically, SSA/Ps are detected when the amount of MUC5AC protein, as measured by the intensity or level of MUC5AC protein expression, is greater than 20% relative to baseline and the amount of TFF1 protein, as measured by the intensity or level TFF1 protein expression, is greater than 25% relative to baseline.

[0054] In still yet another embodiment, SSA/Ps are detected when the amount of MUC5AC protein, as measured by the number of cells expressing MUC5AC, is greater than 30%, greater than 35%, or greater than 40% relative to baseline and the amount of TFF1 protein, as measured by the intensity or level TFF1 protein expression, is greater than 25%, greater than 30%, or greater than 35% relative to baseline.

Specifically, SSA/Ps are detected when the amount of MUC5AC protein, as measured by the number of cells expressing MUC5AC, is greater than 30% relative to baseline and the amount of TFF1 protein, as measured by the intensity or level TFF1 protein expression, is greater than 25% relative to baseline.

[0055] In a different embodiment, SSA/Ps are detected when the amount of MUC5AC protein, as measured by the intensity or level of MUC5AC protein expression, is greater than 20%, greater than 25%, or greater than 30% relative to baseline and the amount of TFF1 protein, as measured by the number of cells expression TFF1 , is greater than 45%, greater than 50%, or greater than 55% relative to baseline. Specifically, SSA/Ps are detected when the amount of MUC5AC protein, as measured by the intensity or level of MUC5AC protein expression, is greater than 20% relative to baseline and the amount of TFF1 protein, as measured by the number of cells expressing TFF1 , is greater than 45% relative to baseline.

[0056] Alternatively, the amount of MUC5AC and TFF1 protein measured in the sample as compared to a reference value may be used to detect SSA/Ps. As described above, the amount of MUC5AC and TFF1 protein may include the number of cells expressing MUC5AC and TFF1 in the biological sample and/or the intensity or level of MUC5AC and TFF1 protein expression in the biological sample.

[0057] In one embodiment, the amount of MUC5AC and TFF1 protein can be compared using the ratio of the amount of MUC5AC and TFF1 protein in the biological sample as compared with the reference value. For example, a protein is differentially expressed if the ratio of the amount of MUC5AC and TFF1 protein in the biological sample as compared with the reference value is greater than or less than 1 .0. For example, a ratio of greater than 1 , 1.2, 1 .5, 1 .7, 2, 3, 3, 5, 10, 15, 20 or more, or a ratio less than 1 , 0.8, 0.6, 0.4, 0.2, 0.1 , 0.05, 0.001 or less. In a specific embodiment, SSA/Ps are detected when the ratio of the amount of MUC5AC and TFF1 protein in the biological sample as compared with the reference value is greater than 1 .0. In another embodiment, the increase or decrease in amount is measured using p-value. For instance, when using p-value, MUC5AC and TFF1 protein is identified as being differentially expressed between MUC5AC and TFF1 protein in a biological sample and the reference value when the p-value is less than 0.1 , preferably less than 0.05, more preferably less than 0.01 , even more preferably less than 0.005, the most preferably less than 0.001 . In a specific embodiment, SSA/Ps are detected when MUC5AC and TFF1 protein is increased in a biological sample relative to the reference value and the p-value is less than 0.1 , preferably less than 0.05, more preferably less than 0.01 , even more preferably less than 0.005, the most preferably less than 0.001 . In another embodiment, the increase or decrease in amount is measured using fold change. For example, MUC5AC and TFF1 protein may be increased or decreased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, at least 10-fold, at least 1 1 -fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, or at least 75-fold, relative to the reference value. In a specific embodiment, SSA/Ps are detected when MUC5AC and TFF1 protein is increased in a biological sample relative to the reference value and the fold change is at least 5-fold, at least 10- fold, or at least 15-fold greater than the reference value.

[0058] Any suitable reference value known in the art may be used. For example, a suitable reference value may be the amount of MUC5AC and TFF1 protein in a biological sample obtained from a subject or group of subjects of the same species that has no detectable sessile serrated adenomas/polyps (SSA/Ps). Additionally, a suitable reference value may be the amount of MUC5AC and TFF1 protein in a biological sample from the same subject taken from a non-diseased portion of the colon. Further, a suitable reference value may be the amount of MUC5AC and TFF1 protein in a biological sample obtained from a subject or group of subjects of the same species that has healthy colonic tissue. Using such a reference value, SSA/Ps may be detected when the amount of MUC5AC and TFF1 is at least 12-fold greater than the reference value. Alternatively, using such a reference value, SSA/Ps may be detected when the expression level of MUC5AC and TFF1 is at least 15-fold greater than the reference value. For example, using such a reference value, SSA/Ps may be detected when the amount of MUC5AC and TFF1 is at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold greater than the reference value. Further, using such a reference value, SSA/Ps may be detected when the amount of MUC5AC and TFF1 is significantly greater than the reference value.

[0059] In another example, a suitable reference value may be the amount of MUC5AC and TFF1 protein in a biological sample obtained from a subject or group of subjects of the same species that has hyperplastic polyps (HPs). Using such a reference value, SSA/Ps may be detected when the amount of MUC5AC and TFF1 is at least 1.2-fold greater than the reference value. Alternatively, using such a reference value, SSA/Ps may be detected when the amount of MUC5AC and TFF1 is at least 1 .4- fold greater than the reference value. For example, using such a reference value, SSA/Ps may be detected when the amount of MUC5AC and TFF1 is at least 1.2-fold, at least 1.3-fold, at least 1 .4-fold, at least 1 .5-fold, at least 1 .6-fold, at least 1 .7-fold, at least 1.8-fold, at least 1 .9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 1 1 -fold, at least 12-fold, at least 13-fold, at least 14-fold, or at least 15-fold greater than the reference value. Further, using such a reference value, SSA/Ps may be detected when the amount of MUC5AC and TFF1 is significantly greater than the reference value.

[0060] In another example, a suitable reference value may be the amount of MUC5AC and TFF1 protein in a biological sample obtained from a subject or group of subjects of the same species that has sessile serrated adenomas/polyps (SSA/Ps) as measured via standard methods. Using such a reference value, SSA/Ps may be detected when the amount of MUC5AC and TFF1 is less than 5-fold different than the reference value. Alternatively, using such a reference value, SSA/Ps may be detected when the amount of MUC5AC and TFF1 is less than 2-fold different than the reference value. Further, using such a reference value, SSA/Ps may be detected when the amount of MUC5AC and TFF1 is not significantly different than the reference value.

[0061 ] In another example, a suitable reference value may be a

measurement of the amount of MUC5AC and TFF1 protein in a reference sample obtained from the same subject. The reference sample comprises the same type of biological fluid as the test sample, and may or may not be obtained from the subject when SSA/Ps were not suspected. A skilled artisan will appreciate that it is not always possible or desirable to obtain a reference sample from a subject when the subject is otherwise healthy. For example, in an acute setting, a reference sample may be the first sample obtained from the subject at presentation. In another example, when monitoring the effectiveness of a therapy, a reference sample may be a sample obtained from a subject before therapy began. In such an example, a subject may have suspected SSA/Ps but may not have other symptoms of SSA/Ps or the subject may have suspected SSA/Ps and one or more other symptom of SSA/Ps.

[0062] In addition to the amount of MUC5AC and TFF1 protein, MUC5AC and TFF1 protein expression pattern in the biological sample may be used to detect SSA/Ps. In one embodiment, co-localization of MUC5AC and TFF1 protein is used to detect SSA/Ps. As used herein, "co-localized" or "co-localization" refers to observation of the spatial overlap between two different fluorescent labels, each having a separate emission wavelength. SSA/Ps are detected when MUC5AC and TFF1 co-localization is greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95%. Specifically, SSA/Ps are detected when MUC5AC and TFF1 co- localization is greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0063] In another embodiment, localization of MUC5AC and TFF1 protein expression may be used to detect SSA/Ps. MUC5AC and TFF1 protein expression may be throughout the polyp or only on the luminal surface of the polyp. SSA/Ps are detected when MUC5AC and TFF1 protein expression pattern is throughout the polyp. More specifically, MUC5AC and TFF1 protein expression is detectable along the entire length of the architecturally compromised crypts of the polyp. Further, MUC5AC and TFF1 protein expression is detectable along the entire length of the architecturally compromised crypts of the polyp including the base.

(d) classifying a subject

[0064] A subject may be classified based on the amount and/or pattern of MUC5AC and TFF1 protein measured in the sample. Classifying a subject based on the amount and/or pattern of MUC5AC and TFF1 protein measured in a biological sample obtained from the subject may be used to detect SSA/Ps, differentiate SSA/Ps from HPs, determine treatment for serrated polyps, to monitor the progression of serrated polyps and/or to monitor the response to treatment for serrated polyps. Generally speaking, a subject may be classified as having an increased or decreased amount of MUC5AC and TFF1 protein. In some embodiments, the amount of MUC5AC and TFF1 protein may be compared to a reference value, wherein an increased amount of MUC5AC and TFF1 protein is an amount above the reference value and a decreased amount is an amount equal to or below the reference value. Additionally, a subject may be classified as having co-localized or non-co-localized MUC5AC and TFF1 protein and/or MUC5AC and TFF1 protein throughout the polyp or only on the luminal surface of the polyp.

[0065] The determination of SSA/Ps may be used to select treatment for subjects. As explained herein, MUC5AC and TFF1 protein can classify a subject as having HPs or SSA/Ps and into groups that might benefit from more aggressive therapy or determine the appropriate treatment for the subject. In an embodiment, a subject classified as having SSA/Ps may be treated. A skilled artisan would be able to

determine standard treatment for SSA/Ps. Accordingly, the methods disclosed herein may be used to select treatment for serrated polyp subjects. In an embodiment, the subject is treated based on the expression level and pattern of MUC5AC and TFF1 protein measured in the sample. This classification may be used to identify groups that are in need of treatment or not or in need of more aggressive treatment. The term "treatment" or "therapy" as used herein means any treatment suitable for the treatment of SSA/Ps. Treatment may consist of standard treatments for SSA/Ps. Non-limiting examples of standard treatment for SSA/Ps include include polypectomy, endoscopic resection, and surgical resection, all followed with surveillance. Additionally, the treatment decision may be made based on evidence of progression from SSA/Ps to cancer.

[0066] In any of the foregoing embodiments, the method may further comprises determining an expression level of at least one gene selected from MUC17, VSIG1 , CTSE, TFF2, TM4SF4, SERPINB5, KLK7, REG4, SLC6A14, ANXA10, HTR1 D, KLK1 1 , DUOXA2, VNN1 , SULT1 C2, AQP5, PI3, CLDN1 , DUSP4, SLC6A20, TRIM29, PRSS22, TACSTD2, ST3GAL4, SDR16C5, ALDOB, HOXB13, KRT7, GJB4, APOB, PSCA, CIDEC, XKR9, DPCR1 , RAB3B, FIBCD1 , NXF3, PDZK1 IP1 , ZIC5, CEACAM18, CXCL1 , MDFI, ONECUT2, SLC37A2, FAM3B, B4GALNT2, POPDC3, SLC30A10, PCDH20, UGT2A3, HSD3B2, CNTFR, EYA2, PITX2, G6PC, UGT1A4, PRKG2,

ADH1 C, CWH43, SLC17A8, MOCS1 , NPY1 R, TRIM9, and TMIGD1 . An increase in the expression level at least one of MUC17, VSIG1 , CTSE, TFF2, TM4SF4, SERPINB5, KLK7, REG4, SLC6A14, ANXA10, HTR1 D, KLK1 1 , DUOXA2, VNN1 , SULT1 C2, AQP5, PI3, CLDN1 , DUSP4, SLC6A20, TRIM29, PRSS22, TACSTD2, ST3GAL4, SDR16C5, ALDOB, HOXB13, KRT7, GJB4, APOB, PSCA, CIDEC, XKR9, DPCR1 , RAB3B, FIBCD1 , NXF3, PDZK1 IP1 , ZIC5, CEACAM18, CXCL1 , MDFI, and ONECUT2 relative to a reference value may indicate SSA/Ps. A decrease in the expression level of at least one of SLC37A2, FAM3B, B4GALNT2, POPDC3, SLC30A10, PCDH20, UGT2A3, HSD3B2, CNTFR, EYA2, PITX2, G6PC, UGT1A4, PRKG2, ADH1 C, CWH43,

SLC17A8, MOCS1 , NPY1 R, TRIM9, and TMIGD1 relative to the control value may indicate SSA/Ps.

EXAMPLES

[0067] The following examples are included to demonstrate various embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Introduction for the Examples.

[0068] Colon cancer is the second largest cause of cancer-related deaths in the United States. Colonic neoplasms originate primarily from colon polyps, and develop via partially overlapping but mechanistically distinct pathways that have been designated as the adenomatous and serrated pathways. About 60% of colon cancers are thought to originate from adenomas via "suppressor" or "mutator" pathways, involving APC mutations in combination with constitutive stimulation of RAS-RAF-MAPK signaling due to gain-of-function mutations in BRAF or KRAS. Accumulating evidence indicates that the majority of other colon adenocarcinomas, possibly 20-30%, arise from a subset of serrated polyps, designated sessile serrated adenomas/polyps (SSA/Ps), which were previously classified as hyperplastic polyps and thought to have little or no tumorigenic potential. [0069] The "serrated pathway" is believed to be responsible for the progression of SSA/Ps to colonic adenocarcinoma. In addition, SSA/Ps exhibit a high frequency of gain-of-function mutations in BRAF gene ranging from 70-100%; BRAF mutations have been implicated in a variety of cancers due to their activation of the MAPK signaling pathway.

[0070] The current pathologic classification of serrated polyps includes hyperplastic polyps (HPs), traditional serrated adenomas (TSA) and sessile serrated adenomas/polyps (SSA/Ps) with the latter displaying the strongest association with an increased risk for colon cancer. SSA/Ps are histologically distinct from HPs, and full-length serration of crypts in combination with lateral dilatation or "boot" shaped deformities at the crypt bases as well as "reverse maturation," or the presence of mature goblet cells and/or foveolar-type cells at the base.

[0071 ] Nevertheless, differentiating between SSA/Ps and HPs on routine histological examination can be challenging, particularly in small or fragmented samples. This has been highlighted by a number of studies documenting the frequent misclassification of SSA/Ps as HPs, resulting in inadequate follow-up. Conversely, misclassifying an HP as an SSA/P may result in unnecessary cancer screening in these patients. A reliable diagnostic test that may help in this distinction would be very useful in identifying SSA/Ps so that appropriate follow up and screening could be provided to the large number of patients with serrated polyps.

[0072] In the present study, we evaluated the expression of MUC5AC and TFF1 by immunofluorescence and immunoperoxidase staining of SSA/Ps and HPs to determine their possible suitability as diagnostic markers for SSA/Ps.

Example 1. MUC5AC and TFF1 expression in HPs and SSA/Ps.

[0073] With the exception of rare epithelial cells located near the luminal surface that expressed TFF1 or MUC5AC (FIG. 1 ), all 30 normal colon controls had negligible immunofluorescence staining for MUC5AC and TFF1 . Immunoperoxidase staining showed similar results (FIG. 1 ). In contrast, HPs exhibited significantly stronger immunopositivity for both MUC5AC and TFF1 in multiple cells (1 1 .3-fold vs. control, p < 10^"6 and 1 1 .4-fold vs. control, p < 10^"6, respectively) (FIG. 1, FIG. 2). However, these cells were almost never observed at the base of crypts, but rather were confined to the luminal surface of the polyps. SSA/Ps displayed the highest expression of MUC5AC and TFFI by immunostaining (17.5-fold vs. control, p < 10^"9 for MUC5AC and 16.3-fold vs. control, p < 10^"9 for TFF1 ), which often involved the entire length of the crypt including the base. SSA/Ps displayed on average 1 .6 (MUC5AC, p < 0.008) and 1 .4-fold (TFF1 , p < 0.03) higher signal intensity for these markers than HPs. The differences in staining between SSA/Ps and HPs for both proteins were statistically significant (p < 0.008 for MUC5AC and p < 0.03 for TFF1 ). Immunoperoxidase staining of MUC5AC and TFF1 in SSA/Ps and HPs showed similar results to immunofluorescence staining (FIG. 1 )

Example 2. Compared to HPs, SSA/Ps exhibit stronger co-localization of MUC5AC and TFF1.

[0074] In terms of co-localized immunostaining for MUC5AC and TFF1 , HPs showed heterogeneous staining with a range of phenotypes (FIG. 3A). The majority of HPs showed some cells with co-localization of MUC5AC and TFF1 , with two exceptions of 46 HPs analyzed showing more co-localization (FIG. 3A). In contrast, SSA/Ps almost uniformly demonstrated strong, intense co-localization of MUC5AC and TFF1 immunostaining (FIG. 3B). Based on scoring by two Gl pathologists (see

Methods), the degree of co-localization of MUC5AC and TFF1 reached roughly 25% in HPs, whereas co-localization in SSA/P samples approached 57% (p < 0.008) (FIG. 2). Regardless of the degree of co-localization, the expressions of MUC5AC and TFF1 significantly correlated (r² = 0.48, p = 0.0007).

Example 3. Expression of MUC5AC and TFF1 in SSA/Ps is confined to serrated crypts.

[0075] SSA/P samples displayed dramatic differences between serrated crypts and crypts with a normal morphology within the same specimen (not shown). The latter were indistinguishable from normal colonic mucosa by MUC5AC and TFF1 immunostaining, even when they were immediately adjacent to dilated, serrated crypts characteristic of SSA/Ps. The apparent absence of a field effect indicates that, regardless of the underlying cause of MUC5AC and TFF1 over-expression in SSP/As, this is a highly localized change in gene expression confined only to serrated crypts.

Discussion for the Examples.

[0076] Due to the inherent difficulties in distinguishing between HPs and SSP/As that are encountered in the routine practice of pathology, identifying clinically applicable biomarkers that distinguish between SSA/Ps and HPs has been an area of ongoing research for some time. In this study, we have shown that MUC5AC and TFF1 , genes commonly expressed in gastric mucosa, are found in both HPs and SSA/Ps, but differ in their distribution, and co-expression. The different expression properties of MUC5AC and TFF1 in SSA/Ps and HPs may serve as clinically useful diagnostic criteria.

[0077] MUC5AC belongs to a family of secreted mucins found primarily in the mucosa of therespiratory tract, stomach, and reproductive organs. Trefoil factors are a 3-member family of small secreted proteins involved in the repair of mucosal damage due to their ability to inhibit apoptosis and stimulate cell migration, proliferation and angiogenesis. The present study revealed significant differences in expression of MUC5AC and TFF1 in SSA/Ps and HPs. Although previous reports have observed over-expression of these proteins in the SSA/Ps the analysis presented here provides evidence that both the level and the cellular pattern of expression is distinctly different in SSA/Ps as compared to HPs. We observed a significant increase in not only the intensity of immunostaining but also in the percentage of cells expressing MUC5AC and TFF1 in SSA/Ps.

[0078] Based on both the literature and daily clinical practice, pathologists are aware that many serrated polyps cannot be reliably classified based on morphology alone, which in turn affects the clinician's ability to assign appropriate follow-up to many patients with serrated polyps. Our findings suggest that expression of TFF1 and

MUC5AC as measured by immunostaining are capable of distinguishing potentially tumorigenic polyps among serrated polyps, and could aid in the distinction between SSA/Ps and HPs in histologically suboptimal samples. Methods for the Examples.

[0079] Sample selection and pathologic examination: A total of 37 SSA/Ps, 46 HPs, and 30 normal colon were retrieved from the archives of the University of Arkansas for Medical Sciences and Central Arkansas Veteran Healthcare Center. All specimens were biopsies obtained by colonoscopy, and were formalin-fixed and embedded in paraffin. All specimens were reviewed and the diagnoses confirmed by two pathologists with expertise in Gl pathology (LKK and LWL), and the size and location of the polyps were recorded. Because this is the initial study in determining the utility of these markers, we limited our analysis to morphologically unequivocal HPs from the left colon and SSA/Ps found in the right colon.

[0080] Immunofluorescence and Immunohistochemistry: Antibodies for MUC5AC (Cat. #MA512178) and TFF1 (Cat. #PA128875) were purchased from Fisher Scientific (Hanover Park, IL). Four-micron sections of formalin-fixed paraffin-embedded tissue (FFPE) were mounted on positively charged super-frost/plus slides. Section were deparaffinized with Neo-ClearH Xylene Substitute (Millipore Cat. #65351 ) and

rehydrated using graded series of alcohol to phosphate-buffered saline (PBS). Antigen retrieval was performed by incubating slides in 10 mM citrate buffer (pH 6.0) in water bath at 95°C for 30 min followed by incubation at room temperature for another 30 min. Tissue sections were treated with blocking buffer (1 % BSA, 0.012% saponin in PBS) for 30 minutes, incubated with primary antibodies (1 : 100 dilution in blocking buffer) for 2 hours at RT in a humidity chamber and washed in PBS-PBST-PBST (10 minutes each). Immunofluorescence analysis samples were incubated with the appropriate DyLight conjugated secondary antibody (Molecular probes, Grand Island, NY) at dilutions 1 : 1000 for one hour at 37°C in humidity chamber following the manufacturer's

recommendations. After three consecutive 10 minute washes with PBS, PBST and PBS coverslips were mounted using Prolong Diamond antifade mountant with DAPI

(Molecular Probes, Grand Island, NY) and imaged by fluorescent microscopy.

[0081 ] For peroxidase immunohistochemistry (IHC) analysis,

deparaffinized four-micron sections were pre-incubated with a 2.5% normal horse serum blocking solution (Vector laboratories, cat# S-2012) for 30 minutes at room temperature and incubated with primary antibodies for one hour at room temperature. Samples were washed with PBS (phosphate-buffered saline) and PBS with 1 % Tween 20. Peroxidase immunostaining was performed, after treatment with BLOXALL™ (Vector Laboratories) endogenous peroxidase blocking solution, using the ImmPRESS polymer system and ImmPACT DAB substrate (Vector Laboratories) per the

manufacturer's instructions. Controls included no primary antibody.

[0082] Image analysis: Image analysis was performed using ImageJ software (NIH). The mean intensities of signals on RGB split images corrected for background were measured in at least 3 representative crypts per sample and averaged. In order to quantify the number of cells with sufficient co-localization we utilized a program "Intensity Correlation Analysis", located within the co-localization plugins for ImageJ in order to generate gradient intensive images. The number of cells was counted from each subsequent image; MUC5AC (green), TFF1 (red), and colocalization (yellow). The co-expression was determined as a percentage of cells with colocalized signals for MUC5AC and TFF1 . In addition to software-based analysis, expression and co-localization were scored by two Gl pathologists (LKK and GHE, reviewed by LWL) based on the percentage of serrated crypt cells staining (0: none; 1 : 1 -25%; 2: 26-50%; 3: 51 -75%; 4: >76%) and intensity of staining (0-4).

[0083] Statistical analysis: Data are presented as means ± standard deviation (SD). The statistical analysis was performed with SPSS 1 1 .5 software.

Multiple comparisons were analyzed by one-way ANOVA. A p value < 0.05 was considered significant.

References for the Examples.

1 . U.S. Cancer Statistics Working Group. United States Cancer Statistics: 1999-201 1

Incidence and Mortality Web-based Report. Atlanta: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention and National Cancer Institute; 2014. Available at: www.cdc.gov/uscs.

2. Leggett B, Whitehall V. Role of the serrated pathway in colorectal cancer

pathogenesis. Gastroenterology 2010; 138:2088-2100. nover DC. Update on the serrated pathway to colorectal carcinoma. Hum Pathol 201 1 ;42: 1 -10.

orlakovic E, Skovlund E, Snover DC, Torlakovic G, Nesland JM. Morphologic reappraisal of serrated colorectal polyps. Am J Surg Pathol 2003;27:65-81 .

akinen M.J. Colorectal serrated adenocarcinoma. Histopathology 2007;50: 131-150. 'Brien MJ. Hyperplastic and serrated polyps of the colorectum. Gastroenterol Clin

North Am 2007;36:947-968.

'Brien MJ, Zhao Q, Yang S. Colorectal serrated pathway cancers and precursors.

Histopathology 2015;66:49-65.

ex DK, Ahnen DJ, Baron JA, et al. Serrated lesions of the colorectum: review and recommendations from an expert panel. Am J Gastroenterol 2012; 107: 1315-29. ang S, Mack C, et al. Comparison of Microsatellite Instability, CpG Island

Methylation Phenotype, BRAF and KRAS Status in Serrated Polyps and Traditional Adenomas Indicates Separate Pathways to Distinct Colorectal Carcinoma End Points. American Journal of Surgical Pathology 2006;30: 1491 -1501 .

Kim KM, Lee EJ, Ha S, et al. Molecular features of colorectal hyperplastic polyps and sessile serrated adenoma/polyps from Korea. Am J Surg Pathol 201 1 ;35: 1274- 86.

Mesteri I, Bayer G, Meyer J, et al. Improved molecular classification of serrated lesions of the colon by immunohistochemical detection of BRAF V600E. Mod Pathol 2014;27: 135-44.

Spring KJ, Zhao ZZ, Karamatic R, et al. High prevalence of sessile serrated adenomas with BRAF mutations: a prospective study of patients undergoing colonoscopy. Gastroenterology 2006; 131 : 1400-7.

Delker DA, McGettigan BM, Kanth P, Pop S, Neklason DW, Bronner MP, Burt RW, Hagedorn CH. RNA sequencing of sessile serrated colon polyps identifies

differentially expressed genes and immunohistochemical markers. PLoS One

2014;9. e88367

Burotto M, Chiou VL, Lee JM, Kohn EC. The MAPK pathway across different malignancies: a new perspective. Cancer 2014; 120:3446-56. Snover DC, Jass JR, Fenoglio-Preiser C, et al. Serrated polyps of the large intestine: a morphologic and molecular review of an evolving concept. Am J Clin Pathol 2005; 124:380-391 .

Wong NA, Hunt LP, Novelli MR, Shepherd NA, Warren BF. Observer agreement in the diagnosis of serrated polyps of the large bowel. Histopathology 2009;55:63-6. Khalid O, Radaideh S, Cummings OW, O'Brien MJ, Goldblum JR, Rex DK.

Reinterpretation of histology of proximal colon polyps called hyperplastic in 2001 . World J Gastroenterol 2009; 15:3767-70.

Slaughter DP, Southwick HW, Smejkal W. Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer 1953;6:963- 8.

Seregni E, Botti C, Massaron S, et al. Structure, function and gene expression of epithelial mucins. Tumori. 1997 ;83:625-32.

Henke MO, John G, Germann M, Lindemann H, Rubin BK. MUC5AC and MUC5B mucins increase in cystic fibrosis airway secretions during pulmonary exacerbation. Am J Respir Crit Care Med 2007;175:816-21.

Ma R, Wang Y, Cheng G, Zhang HZ, Wan HY, Huang SG. MUC5AC expression upregulation goblet cell hyperplasia in the airway of patients with chronic obstructive pulmonary disease. Chin Med Sci J 2005;20:181 -4.

Evans CM, Raclawska DS, Ttofali F, et al. The polymeric mucin Muc5ac is required for allergic airway hyperreactivity. Nat Commun 2015;6:6281 .

Shaoul R, Okada Y, Cutz E, Marcon MA. Colonic expression of MUC2, MUC5AC, and TFF1 in inflammatory bowel disease in children. Journal of pediatric

gastroenterology and nutrition 2004;38:488-93.

Lau SK, Weiss LM, Chu PG. Differential expression of MUC1 , MUC2, and MUC5AC in carcinomas of various sites: an immunohistochemical study. Am J Clin Pathol 2004; 122:61 -9.

Yonezawa S, Higashi M, Yamada N, et al. Mucins in human neoplasms: clinical pathology, gene expression and diagnostic application. Pathol Int 201 1 ;61 :697-716. http://proteinatlas.org Kinoshita K, Taupin DR, Itoh H, Podolsky DK. Distinct pathways of cell migration and antiapoptotic response to epithelial injury: structure-function analysis of human intestinal trefoil factor. Mol Cell Biol 2000;20:4680-90.39.

Aamann L, Vestergaard EM, Gronbak H. Trefoil factors in inflammatory bowel disease. World J Gastroenterol 2014;20:3223-30.

Rio MC, Bellocq JP, Daniel JY, et al. Breast cancer-associated pS2 protein:

synthesis and secretion by normal stomach mucosa. Science 1988;241 :705-8.

Longman RJ, Douthwaite J, Sylvester PA, et al. Coordinated localisation of mucins and trefoil peptides in the ulcer associated cell lineage and the gastrointestinal mucosa. Gut 2000;47:792-800.

Im S, Yoo C, Jung JH, Choi HJ, Yoo J, Kang CS. Reduced expression of TFF1 and increased expression of TFF3 in gastric cancer: correlation with clinicopathological parameters and prognosis. Int J Med Sci 2013; 10: 133-40.

Feng G, Zhang Y, Yuan H, et al. DNA methylation of trefoil factor 1 (TFF1 ) is associated with the tumorigenesis of gastric carcinoma. Mol Med Rep 2014;9:109- 17.

Arumugam T, Brandt W, Ramachandran V, et al. Trefoil Factor 1 Stimulates Both Pancreatic Cancer and Stellate Cells and Increases Metastasis. Pancreas

201 1 ;40:815-822.

Van Seuningen I, Pigny P, Perrais M, Porchet N, Aubert JP. Transcriptional regulation of the 1 1 p15 mucin genes towards new biological tools in human therapy, in inflammatory diseases and cancer? Front Biosci 2001 ;6: 1216-34.

Renaud F, Vincent A, Mariette C et al. MUC5AC hypomethylation is a predictor of microsatellite instability independently of clinical factors associated with colorectal cancer. Int J Cancer 2014; doi: 10.1002/ijc.29342. [Epub ahead of print]

Chen Y, Garvin LM, Nickola TJ et al. IL-1 β induction of MUC5AC gene expression is mediated by CREB and NF-κΒ and repressed by dexamethasone. Am J Physiol Lung Cell Mol Physiol 2014;306:L797-807. Jonckheere N, Van Der Sluis M, Velghe A et al. Transcriptional activation of the murine Muc5ac mucin gene in epithelial cancer cells by TGF-beta/Smad4 signalling pathway is potentiated by Sp1 . Biochem J 2004;377:797-808.

Young HWJ, Williams OW, Chandra D et al. Central Role of Muc5ac Expression in Mucous Metaplasia and Its Regulation by Conserved 5' Elements. Am J Resp Cell Mol Biol 2007;37: 273-290.

Park ET, Gum JR, Kakar S et al. Aberrant expression of SOX2 upregulates

MUC5AC gastric foveolar mucin in mucinous cancers of the colorectum and related lesions. Int J Cancer 2008; 122: 1253-60.

Inaguma S, Kasai K, Ikeda H. GLI1 facilitates the migration and invasion of pancreatic cancer cells through MUC5AC-mediated attenuation of E-cadherin.

Oncogene 201 1 ; 30:714-23.

Al-azzeh ED, Fegert P, Blin N, Gott P. Transcription factor GATA-6 activates expression of gastroprotective trefoil genes TFF1 and TFF2. Biochim Biophys Acta 2000; 1490:324-32.

Sun JM1 , Spencer VA, Li L et al. Estrogen regulation of trefoil factor 1 expression by estrogen receptor alpha and Sp proteins. Exp Cell Res. 2005;302:96-107.

Shin IS, Park JW, Shin NR et al. Melatonin inhibits MUC5AC production via suppression of MAPK signaling in human airway epithelial cells. J Pineal Res.

2014;56:398-407.

Khan P, Drobic B, Perez-Cadahia B, Healy S, He S, Davie JR. Mitogen- and stressactivated protein kinases 1 and 2 are required for maximal trefoil factor 1 induction. PLoS One 2013;8:e63189.

Inaguma S, Kasai K, Ikeda H. GLI1 facilitates the migration and invasion of pancreatic cancer cells through MUC5AC-mediated attenuation of E-cadherin.

Oncogene 201 1 ; 30:714-23.

Hoshi H, Sawada T, Uchida M et al. Tumor-associated MUC5AC stimulates in vivo tumorigenicity of human pancreatic cancer. Int J Oncol 201 1 ;38:619-27. Rodrigues S, Rodrigue CM, Attoub S et al. Induction of the adenoma-carcinoma progression and Cdc25A-B phosphatases by the trefoil factor TFF1 in human colon epithelial cells. Oncogene 2006;25:6628-36.

Bougen NM, Amiry N, Yuan Y et al. Trefoil factor 1 suppression of E-CADHERIN enhances prostate carcinoma cell invasiveness and metastasis. Cancer Lett 2013;332: 19-29.

Rodrigues S, Attoub S, Nguyen QD et al. Selective abrogation of the proinvasive activity of the trefoil peptides pS2 and spasmolytic polypeptide by disruption of the EGF receptor signaling pathways in kidney and colonic cancer cells. Oncogene 2003;22:4488-97.

Bartman AE, Sanderson SJ, Ewing SL et al. Aberrant expression of MUC5AC and MUC6 gastric mucin genes in colorectal polyps. Int J Cancer 1999;80:210-8.

Biemer-Huttmann AE, Walsh MD, McGuckin MA et al. Immunohistochemical staining patterns of MUC1 , MUC2, MUC4, and MUC5AC mucins in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum. J

Histochem Cytochem 1999;47: 1039- Percinel S, Savas B, Ensari A et al. Mucins in the colorectal neoplastic spectrum with reference to conventional and serrated adenomas. Turk J Gastroenterol 2007; 18:230-8.

Mochizuka A, Uehara T, Nakamura T, Kobayashi Y, Ota H. Hyperplastic polyps and sessile serrated 'adenomas' of the colon and rectum display gastric pyloric differentiation. Histochem Cell Biol 2007;128:445-55.

Walsh MD, Clendenning M, Williamson E et al. Expression of MUC2, MUC5AC, MUC5B, and MUC6 mucins in colorectal cancers and their association with the CpG island methylator phenotype. Mod Pathol 2013;26: 1642-56.

Claims

CLAIMS What is claimed is:

1 . A method of detecting sessile serrated adenomas/polyps (SSA/Ps) in a subject, the method comprising:

a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; and b) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is greater than 20% relative to baseline and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp.

2. A method of differentiating sessile serrated adenomas/polyps (SSA/Ps) from hyperplastic polyps (HPs) in a subject, the method comprising:

a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; and b) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is greater than 20% relative to baseline and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp.

3. A method of predicting the likelihood that a colorectal polyp in a subject will develop into colorectal cancer, the method comprising:

a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; and b) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is greater than 20% relative to baseline and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp,

wherein the detection of SSA/Ps in the subject indicates an increased likelihood of developing colorectal cancer.

4. The method of any of claim 1 -3, wherein the amount of MUC5AC and TFF1 protein is measured by the number of cells expressing MUC5AC and TFF1 .

5. The method of claim 4, wherein the baseline is the number of cells in the

biological sample.

6. The method of claim 4, wherein the amount of MUC5AC protein is greater than 30% and the amount of TFF1 protein is greater than 45% relative to baseline.

7. The method of any of claim 1 -3, wherein the amount of MUC5AC and TFF1 protein is measured by the intensity or level of MUC5AC and TFF1 protein expression.

8. The method of claim 7, wherein the baseline is the background staining in the biological sample.

9. The method of claim 7, wherein the amount of MUC5AC protein is greater than 20% and the amount of TFF1 protein is greater than 25% relative to baseline.

10. A method of detecting sessile serrated adenomas/polyps (SSA/Ps) in a subject, the method comprising:

a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject;

b) comparing the amount of MUC5AC and TFF1 protein in the biological sample to a reference value; and

c) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is significantly increased relative to the reference value and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp.

1 1 . A method of differentiating sessile serrated adenomas/polyps (SSA/Ps) from hyperplastic polyps (HPs) in a subject, the method comprising: a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; b) comparing the amount of MUC5AC and TFF1 protein in the biological sample to a reference value; and

c) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is significantly increased relative to the reference value and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp.

12. A method of predicting the likelihood that a colorectal polyp in a subject will develop into colorectal cancer, the method comprising:

a) determining the amount and/or pattern of MUC5AC and TFF1 protein in a biological sample obtained from a colorectal polyp of the subject; b) comparing the amount of MUC5AC and TFF1 protein in the biological sample to a reference value; and

c) detecting SSA/Ps in the subject when the amount of MUC5AC and TFF1 protein is significantly increased relative to the reference value and/or the expression pattern of MUC5AC and TFF1 protein is co-localized and detectable throughout the polyp,

wherein the detection of SSA/Ps in the subject indicates an increased likelihood of developing colorectal cancer.

13. The method of any one of the preceding claims, wherein the co-localization is greater than 25%.

14. The method of claim 13, wherein the co-localization is greater than 30%.

15. The method of claim 13, wherein the co-localization is greater than 40%.

16. The method of claim 13, wherein the co-localization is greater than 50%.

17. The method of any one of the preceding claims, wherein the MUC5AC and TFF1 are detectable along the entire length of the architecturally compromised crypts of the polyp.

18. The method of any one of claims 10-12, wherein the amount of MUC5AC and TFF1 is at least 12-fold greater than the reference value when the reference value is healthy colonic tissue.

19. The method of any one of claims 10-12, wherein the amount of MUC5AC and TFF1 is at least 15-fold greater than the reference value when the reference value is healthy colonic tissue.

20. The method of any one of claims 10-12, wherein the amount of MUC5AC and TFF1 is at least 1.2-fold greater than the reference value when the reference value is hyperplastic polyp tissue.

21 . The method of any one of claims 10-12, wherein the amount of MUC5AC and TFF1 is at least 1.4-fold greater than the reference value when the reference value is hyperplastic polyp tissue.

22. The method of any one of the preceding claims, wherein the subject is treated based on the detection of SSA/Ps.

23. The method of any one of the preceding claims, wherein the subject is subjected to an increased frequency of colonoscopies based on the detection of SSA/Ps.

24. The method of any one of the preceding claims, wherein both amount and

pattern are used to detect SSA/Ps.

25. The method of any one of the preceding claims, wherein the biological sample is biopsied tissue.

26. The method of any one of the preceding claims, wherein the amount of MUC5AC is determined using an antibody specific to MUC5AC and the amount of TFF1 is determined using an antibody specific to TFF1 .

27. The method of claim 26, wherein the amount of MUC5AC and TFF1 is

determined using an antibody specific to MUC5AC and the amount of TFF1 is determined using an antibody specific to TFF1 , and secondary antibodies conjugated to fluorophores, wherein a first secondary antibody is specific to MUC5AC antibody and conjugated to a first fluorophore and a second secondary antibody is specific to TFF1 and conjugated to a second fluorophore.

28. The method of claim 26, wherein the amount of MUC5AC and TFF1 is

determined using an antibody specific to MUC5AC and the amount of TFF1 is determined using an antibody specific to TFF1 , and secondary antibodies conjugated to peroxidase, wherein a first secondary antibody is specific to MUC5AC antibody and conjugated to peroxidase and a second secondary antibody is specific to TFF1 and conjugated to peroxidase.