WO2011060098A1

WO2011060098A1 - Methods for predicting post-surgery risk associated with ileal pouch-anal anastomosis

Info

Publication number: WO2011060098A1
Application number: PCT/US2010/056271
Authority: WO
Inventors: Derren Barken; Leonard Eggleston; Sharat Singh
Original assignee: Prometheus Laboratories Inc.
Priority date: 2009-11-10
Filing date: 2010-11-10
Publication date: 2011-05-19

Abstract

The methods of the present invention are useful in predicting or determining a risk of a patient developing dysplasia/cancer and/or inflammatory complications following a surgical procedure whereby the colon is removed and an internal pouch is created. With the present invention, it is possible to predict patients who will have a particular risk of disease complications to pouch surgery such as ileal pouch-anal anastomosis. The marker for a risk of a patient developing dysplasia/cancer is OmpC, the marker for developing a risk of surgical complications is ANCA.

Description

METHODS FOR PREDICTING POST-SURGERY RISK ASSOCIATED WITH ILEAL POUCH- ANAL ANASTOMOSIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 61/259,978, filed November 10, 2009, and U.S. Provisional Application No. 61/334,348, filed May 13, 2010, all of which are hereby incorporated by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] Since the publication of a landmark report suggesting that the finding of dysplasia in a blind mucosal biopsy of the rectum correlated with the presence of invasive cancer elsewhere in the colon (Morson et al , Gut, 8:423-434 ( 1967)), experts have come to advocate serial endoscopic examinations with nontargeted biopsies to exclude the presence of dysplasia as a means of stratifying UC patients by risk status for the development of colorectal cancer. Cancer develops through a chronic inflammation-dysplasia-cancer pathway. Therefore, patients are entered into a surveillance program in order to detect neoplastic alterations (dysplasia) before the diagnosis of cancer (Ullman et al , Inflamm. Bowel. Dis. , 15:630-638 (2009)).

[0003] Ileal pouch-anal anastomosis (IPAA) is a complex surgical procedure developed to avoid a permanent stoma (opening for collecting waste) in cases where the entire colon and rectum needs to be removed. This procedure is most often used to treat patients with chronic ulcerative colitis and inherited syndromes associated with colon cancers {e.g., familial adenomatous polyposis, which carries a nearly 100 percent risk of colon cancer).

[0004] The EPAA procedure involves removal of the entire colon and rectum with preservation of the anus and sphincter muscles. After removal of the colon and rectum, the surgeon constructs a pouch from the end of the small intestine and attaches it directly to the anus. In order to allow time for the pouch to heal, a temporary ileostomy (opening in the front of the abdomen) is created. The ileostomy is reversed a few months later in a second operation and the patient begins to pass bowel movements through the anus, with only slight alterations in the frequency of bowel movements. In most cases, the IPAA procedure can be performed laparoscopically, which minimizes the incision length and decreases postoperative discomfort and hospital stay. [0005] Crohn's disease (CD) of the pouch and pouchitis are the most common long-term inflammatory complications of IPAA. Other inflammatory complications include cuffitis (inflammation in the rectal muscular cuff), proximal small bowel bacterial overgrowth, and irritable pouch syndrome (IPS). See, Yu et al. , World J. Gastroenterol , 13:5598-5604 (2007).

[0006] Reported cumulative frequency rates of pouchitis 10- 1 1 years after IPAA surgery range from 23% to 46% (Penna et ai, Gut, 38:234-239 ( 1996); Fazio et αΙ., Αηη. Surg. , 222: 120-127 (1995)), and approximately 50% of patients who have undergone IPAA surgery for UC will develop at least one episode of pouchitis (Stocchi et ai, Gastroenterol. Clin. North Am. , 30:223-241 (2001 )). The estimated incidence within 12 months after ileostomy was as high as 40% in a European study (Gionchetti et ai , Gastroenterology, 124: 1202- 1209 (2003)). See also, Yu et al , World J. Gastroenterol , 13:5598-5604 (2007).

[0007] Pouchitis almost exclusively occurs in patients with underlying UC and is rarely seen in patients with familial adenomatous polyposis (FAP) (Penna et al, Br. J. Surg., 80:765-767 ( 1993); Tjandra et al , Am. J. Surg. , 165:322-325 ( 1993)). Although the etiology and pathogenesis of pouchitis are not entirely clear, the bulk of evidence points towards an abnormal mucosal immune response (innate and adaptive) to altered microflora in the pouch leading to acute and/or chronic inflammation (Gionchetti et al, Gastroenterology, 124: 1202- 1209 (2003); Sandborn, Gastroenterology, 107: 1856- 1860 (1994); Gosselink et al , Dis. Colon Rectum, 47:876-884 (2004); Gionchetti et al , Gastroenterology, 1 19:305-309 (2000); Mimura et al, Gut, 53: 108-1 14 (2004); Komanduri et al , Clin. Gastroenterol. Hepatol , 5:352-360 (2007)). See also, Yu et al, World J. Gastroenterol , 13:5598-5604 (2007).

[0008] Fleshner et al. (Clin. Gastroenterol. Hepatol , 6:561 -8 (2008)) found that preoperatively both pANCA and anti-CBirl expression were associated with pouchitis after IPAA. Anti-CBirl increases the incidence of AP (acute pouchitis) only in patients who have low-level pANCA expression, and increases the incidence of CP (chronic pouchitis) only in patients who have high-level pANCA expression.

[0009] Hui et al. (Dis. Colon Rectum, 48: 1254- 1262 (2005)) found that indeterminate colitis patients who had a positive antibody reactivity profile before ΓΡΑΑ had a significantly higher incidence of continuous pouch inflammation (clinically known as chronic pouchitis or Crohn's disease) after surgery than those with a negative profile. [0010] Melmed et al. (Dis. Colon Rectum, 5 1 : 100- 108 (2008)) found that patients with UC (ulcerative colitis) and IC (indeterminate colitis) with a family history of CD or preoperative ASCA-IgA seropositivity were more likely to be diagnosed with CD after IPAA.

[0011] Although some progress has been made in identifying risk factors for pouchitis, there is a need in the art for improved methods of determining those individuals at risk of developing dysplasia and/or other complications following a surgical procedure such as IPAA. The present invention satisfies this need and provides related advantages as well.

BRIEF SUMMARY OF THE INVENTION

[0012] The present invention provides prognostic methods for predicting whether a patient will have a particular risk of developing postoperative complications such as dysplasia/cancer and/or one or more inflammatory complications (e.g., pouchitis) following a colectomy with the creation of an internal pouch.

[0013] In one aspect, the present invention provides a method for predicting or determining a risk of developing dysplasia/cancer and/or an inflammatory complication such as pouchitis in an individual following a surgical procedure whereby the colon is removed and an internal pouch is created (e.g., IPAA), the method comprising:

(a) analyzing a sample obtained from the individual to determine the presence, level, or genotype of one or more markers in the sample to obtain a marker profile; and

(b) predicting or determining a risk (e.g., probability) that the individual will develop dysplasia/cancer and/or an inflammatory complication such as pouchitis following the surgical procedure based upon the marker profile.

[0014] In certain embodiments, the present invention provides a method for predicting or determining a risk of developing dysplasia/cancer and/or an inflammatory complication (e.g., pouchitis) in an individual following a surgical procedure whereby the colon is removed and an internal pouch is created (e.g., IPAA), the method comprising:

(a) analyzing a sample obtained from the individual to determine the level or genotype of one or more markers in the sample; and

(b) comparing the level or genotype of each of the markers to a reference level or genotype to predict or determine a risk (e.g., probability) that the individual will develop dysplasia/cancer and/or an inflammatory complication (e.g., pouchitis) following the surgical procedure. [0015] In other embodiments, the present invention provides a method for predicting or determining a risk of developing dysplasia/cancer and/or an inflammatory complication (e.g., pouchitis) in an individual following a surgical procedure whereby the colon is removed and an internal pouch is created (e.g., IPAA), the method comprising:

(b) applying a statistical analysis (e.g., quartile analysis, median analysis, etc.) to the marker profile to predict or determine a risk (e.g., probability) that the individual will develop dysplasia/cancer and/or an inflammatory complication (e.g., pouchitis) following the surgical procedure.

[0016] In particular embodiments, the methods utilize multiple serological, protein, and/or genetic markers to provide physicians with valuable prognostic insight into an individual's risk of developing dysplasia, cancer, and/or an inflammatory complication to pouch surgery (e.g., IPAA).

[0017] The presence, level, and/or genotype of one or more markers (e.g., marker profile) may be determined in a sample obtained from an individual prior to the surgical procedure (e.g., preoperatively) and/or following the surgical procedure (e.g., postoperatively). In particular embodiments, the presence, level, and/or genotype of one or more markers of interest is determined prior to the surgical procedure, e.g., at the time of colectomy. In some embodiments, the surgical procedure whereby the colon is removed and an internal pouch is created comprises an ileal pouch-anal anastomosis (IPAA) procedure. In other embodiments, the inflammatory complication is pouchitis (e.g., acute pouchitis, chronic pouchitis, active pouchitis, refractory pouchitis, and the like), Crohn's disease (CD) of the pouch, cuffitis (e.g., inflammation in the rectal muscular cuff), pouch stricture, pouch sinus, proximal small bowel bacterial overgrowth, irritable pouch syndrome (IPS), anismus, or combinations thereof. In further embodiments, the dysplasia/cancer is cancer of the pouch (e.g., cancer in the ileal pouch mucosa).

[0018] In one particular embodiment, the presence and/or level of one or more markers such as, e.g., ASCA IgG, ASCA IgA, anti-OmpC antibody, and/or anti-CBir- 1 antibody is determined in accordance with the methods of the present invention. In other embodiments, additional and/or alternative markers include, but are not limited to, ANCA ELISA, DNAse sensitive pANCA IFA, and combinations thereof. In further embodiments, the genotype of one or more genomic markers such as, e.g., NOD2/CARD 15 variants (e.g., R702W (SNP8), G908R (SNP12), and 1007fs (SNP 13)) is determined, e.g., alone or in conjunction with one or more serological and/or protein markers of interest. In yet other embodiments, additional markers include, but are not limited to, anti-I2 antibody, EGF, cytokines (e.g., TNF-a, IL-6, IL-8, etc.), defensins such as β-defensin, C-Reactive Protein (CRP), serum amyloid A (SAA), and combinations thereof.

[0019] In other embodiments, the methods and markers described herein can be used to predict whether an individual is appropriate for anti-tumor necrosis factor (TNF) therapies and strategies such as, e.g., chimeric monoclonal antibodies (e.g., infliximab), humanized monoclonal antibodies (e.g., CDP571 and PEGylated CDP870), fully human monoclonals (e.g., adalimumab) antibodies, p75 fusion proteins (e.g., etanercept), p55 soluble receptors (e.g., onercept), small molecules such as MAP kinase inhibitors, and combinations thereof.

[0020] In certain aspects, the methods described herein can predict the probability of response, serve as a guide for selecting an initial therapy, serve as a guide for selecting aggressive or non-aggressive treatment (e.g., at the start of therapy or anytime during a therapeutic regimen), and serve as a guide for changing disease behavior.

[0021] Advantageously, by using a marker profile composed of one or multiple markers (e.g., serological, protein, genetic, etc. ) alone or in conjunction with statistical analysis, the assay methods of the present invention provide prognostic value by identifying patients with a risk of developing dysplasia, cancer, and/or an inflammatory complication to pouch surgery (e.g., IPAA). In certain instances, the methods described herein enable classification and/or differentiation of post-surgery risk into different subgroups, e.g., by differentiating pouchitis from other inflammatory and/or non-inflammatory disorders of the pouch.

[0022] Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 illustrates a disease classification system (DCS) according to one embodiment of the present invention.

[0024] FIG. 2 illustrates an exemplary embodiment of a marker profile of the present invention for predicting or determining an individual's risk of developing dysplasia/cancer and/or inflammatory complications such as pouchitis following a surgical procedure whereby the colon is removed and an internal pouch is created (e.g., IPAA).

[0025] FIG. 3 illustrates an exemplary dataset collected from serum samples, wherein the concentration levels of ANCA, ASCA IgA, ASCA IgG, anti-CBirl , anti-OmpC, C reactive protein (CRP), serum amyloid A (SAA), EGF, and anti-I2, as well as the presence or absence of pANCA (e.g., DNAse sensitive pANCA IF A), were determined.

[0026] FIG. 4A illustrates a gel confirming the expression of the GST-I2 antigen. FIG. 4B illustrates a gel confirming the presence of the GST-I2 antigen in the denatured sample (DEN). FIG. 4C illustrates a gel confirming the presence of the GST-I2 antigen in the filtered sample (FIL).

[0027] FIG. 5 illustrates a graph of a sample standard curve with controls as described in Example 10.

[0028] FIG. 6A illustrates an anti-I2 ELISA which utilizes a monoclonal antibody (McAb) against GST and a refolded GST-I2 antigen. FIG. 6B illustrates an anti-I2 ELISA which utilizes neutravidin and a biotinylated refolded GST-I2 antigen.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

[0029] The present invention is based, in part, upon the surprising discovery that the accuracy of predicting whether a patient will have a particular risk or likelihood of developing postoperative complications following pouch surgery can be substantially improved by detecting the presence, level, or genotype of certain prognostic markers in a biological sample from an individual in need of such surgery.

[0030] In particular, the present invention provides methods and systems for predicting or determining a risk of developing dysplasia/cancer and/or an inflammatory complication such as pouchitis in an individual following a surgical procedure whereby the colon is removed and an internal pouch is created (e.g., IPAA). In some aspects, the methods and systems utilize one or multiple serological, protein, and genetic markers alone or in combination with algorithms to provide physicians valuable prognostic insight. In certain aspects, the methods and systems deliver a patient's projected response to a particular therapy. By using one or multiple markers (e.g., serological, protein, and genomic markers), alone or in conjunction with algorithms, the assays provide prognostic value by identifying patients at risk of post- surgery complications associated with IPAA. In certain other instances, the methods enable the classification and/or differentiation of post-surgery risk into different subgroups, e.g., by differentiating pouchitis from other inflammatory and/or non-inflammatory disorders of the pouch. In other aspects, the use of one or multiple markers (e.g., serological, protein, and genomic markers) provide the ability to distinguish responders from non-responders and guide initial therapeutic options and potential to change disease behavior.

[0031] In certain instances, the methods and systems herein comprise a step having a "transformation" or "machine" associated therewith. For example, the level or concentration of many of the prognostic markers is performed with an ELISA technique. An ELIS A includes transformation of the protein for example an autoantibody, into a conjugate of an autoantibody-antigen, which then can be measured with a labeled secondary antibody. In many instances, the label is an enzyme which transforms a substrate into a detectable product. The detectable product measurement is in many instances performed using a plate reader such as a spectrophotometer. In other instances, genetic markers are determined using various amplification techniques such as PCR. Method steps including amplification such as PCR are transformation of single or double strands of nucleic acid into multiple strands for detection. In many instances, the detection includes the use of a fluorophore, which is performed using a machine such as a fluori meter.

II. Definitions

[0032] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0033] The term "classifying" includes "associating" or "categorizing" a sample or an individual with a disease state or prognosis. In certain instances, "classifying" is based on statistical evidence, empirical evidence, or both. In certain embodiments, the methods and systems of classifying use a so-called training set of samples from individuals with known disease states or prognoses. Once established, the training data set serves as a basis, model, or template against which the features of an unknown sample from an individual are compared, in order to classify the unknown disease state or provide a prognosis of the disease state in the individual. In some instances, "classifying" is akin to diagnosing the disease state and/or differentiating the disease state from another disease state. In other instances, "classifying" is akin to providing a prognosis of the disease state in an individual diagnosed with the disease state. [0034] The term "inflammatory bowel disease" or "IBD" includes gastrointestinal disorders such as, e.g., Crohn's disease (CD), ulcerative colitis (UC), and indeterminate colitis (IC). Inflammatory bowel diseases (e.g., CD, UC, and IC) are distinguished from all other disorders, syndromes, and abnormalities of the gastroenterological tract, including irritable bowel syndrome (IBS). U.S. Patent Publication 20080131439, entitled "Methods of

Diagnosing Inflammatory Bowel Disease" is incorporated herein by reference for all purposes.

[0035] As used herein, a "surgical procedure whereby the colon is removed and an internal pouch is created" is synonymous with "pouch surgery" and includes a surgical procedure that results in total or near-total colectomy and creation of an internal reservoir for maintenance of continence. An internal reservoir can be, for example, an ileal reservoir or ileoanal reservoir. Thus, a surgical procedure whereby colon is removed and an internal pouch is created is, for example, colectomy with ileal pouch-anal anastomosis (IPAA).

[0036] The term "pouchitis" includes a non-specific inflammation of a surgically created reservoir which manifests clinically with diarrhea accompanied by additional variable symptoms such as abdominal cramping, fecal urgency, bleeding, and/or fever. Pouchitis can be diagnosed clinically by an increase of at least three stools per day above the post-operative baseline (Sandborn et al. , Am. J. Gastroenterol. , 90:740-747 (1995), which is incorporated herein by reference in its entirety). Characteristic endoscopic features of pouchitis are granularity, friability, loss of vascular pattern, mucous exudate, and/or ulceration of the pouch. The term "pouchitis" also includes a condition which manifests clinically with diarrhea combined with one or more of the characteristic endoscopic features of pouchitis.

[0037] Pouchitis can develop months or years following pouch surgery. The cumulative frequency of pouchitis increases with time such that 15%, 36%, or 46% of patients develop pouchitis 1 , 5, or 10 years, respectively, after pouch surgery (Penna et al., Gastroenterol , 106:A751 (1994), which is incorporated herein by reference in its entirety). The term "early- onset pouchitis" includes a form of pouchitis that develops within twelve months of a surgical procedure whereby the colon is removed and an internal pouch is created.

[0038] Pouchitis can occur acutely or as a chronic condition. "Acute pouchitis" occurs as a single event or as intermittent relapses with pouchitis-free intervals during which suppressive therapy is not required. In contrast, "chronic pouchitis," which accounts for about 5% of cases following IPAA, is characterized by persistent symptoms of pouchitis despite adequate medical therapy, or by the need for continuous medical suppressive therapy with a prompt recurrence of symptoms if medical therapy is discontinued. Chronic pouchitis can be treatment-responsive, requiring ongoing suppressive therapy, or can be treatment-resistant. As used herein, the term "pouchitis" encompasses early-onset, acute, and chronic pouchitis and includes treatment-responsive and treatment-resistant forms of chronic pouchitis.

[0039] "Dysplasia" includes an abnormality in the maturation of cells within a tissue which generally involves an expansion of immature cells with a corresponding decrease in the number and location of mature cells. Dysplasia is often indicative of an early neoplastic process. The term "dysplasia" is typically used when the cellular abnormality is restricted to the originating tissue, as in the case of an early, in situ neoplasm. In certain embodiments, dysplasia is the earliest form of pre-cancerous lesion that is recognizable in a pap smear or in a biopsy by a pathologist, and can be low grade or high grade. Dysplasia is characterized by one or more (preferably all four) of the following major pathological microscopic changes: (1) anisocytosis (cells of unequal size); (2) poikilocytosis (abnormally shaped cells); (3) hyperchromatism; and/or (4) the presence of mitotic figures (an unusual number of cells which are currently dividing). In particular embodiments, the dysplasia is present in any portion of the gastrointestinal tract, including, but not limited to, the esophagous, stomach, small intestine, colon, rectum, anus, and combinations thereof.

[0040] The term "cancer" includes any member of a class of diseases characterized by the uncontrolled growth of aberrant cells. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre- and post-metastatic cancers. Non-limiting examples of different types of cancer include digestive and gastrointestinal cancers (e.g. , colorectal cancer, gastrointestinal stromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, and small intestine cancer); gastric cancer (e.g. , stomach); breast cancer; lung cancer (e.g., non-small cell lung cancer); esophageal cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; prostate cancer; ovarian cancer; renal cancer (e.g., renal cell carcinoma); cancer of the central nervous system; skin cancer; choriocarcinomas; head and neck cancers; hematological malignancies (e.g., leukemia, lymphoma); osteogenic sarcomas (e.g., Ewing sarcoma); soft tissue sarcomas (e.g., Dermatofibrosarcoma Protuberans (DFSP), rhabdomyosarcoma); other soft tissue

malignancies, and papillary thyroid carcinomas. As used herein, a "tumor" comprises one or more cancerous cells. [0041] The term "sample" includes any biological specimen obtained from an individual. Suitable samples for use in the present invention include, without limitation, whole blood, plasma, serum, saliva, urine, stool, tears, any other bodily fluid, tissue samples (e.g., biopsy), and cellular extracts thereof (e.g., red blood cellular extract). In a preferred embodiment, the sample is a serum sample. The use of samples such as serum, saliva, and urine is well known in the art (see, e.g., Hashida et al , J. Clin. Lab. Anal , 1 1 :267-86 (1997)). One skilled in the art will appreciate that samples such as serum samples can be diluted prior to the analysis of marker levels.

[0042] The term "marker" includes any biochemical marker, serological marker, genetic marker, or other clinical or echographic characteristic that can be used in the methods of the present invention. Non-limiting examples of such markers include serological markers such as an anti-neutrophil antibody, an anti-Saccharomyces cerevisiae antibody, an antimicrobial antibody, an acute phase protein, an apolipoprotein, a defensin, a growth factor, a cytokine, a cadherin, a cellular adhesion molecule; genetic markers such as, e.g., NOD2/CARD15; and combinations thereof. In some embodiments, the markers are utilized in combination with a statistical analysis to provide a risk of developing dysplasia, cancer, and/or an inflammatory complication to pouch surgery in an individual.

[0043] The term "marker profile" includes one, two, three, four, five, six, seven, eight, nine, ten, or more diagnostic and/or prognostic marker(s), wherein the markers can be a serological marker, a protein marker, a genetic marker, and the like. In some embodiments, the marker profile together with a statistical analysis can provide physicians and caregivers valuable diagnostic and prognostic insight. In other embodiments, the marker profile with optionally a statistical analysis provides a projected response to biological therapy. A preferred statistical analysis is a quartile score and the quartile score for each of the markers can be summed to generate a quartile sum score. By using one or more markers (e.g., serological, protein, genetic, etc.) in conjunction with statistical analyses, the assays described herein provide diagnostic, prognostic, and therapeutic value by predicting a risk (e.g., probability, likelihood, etc.) of developing dysplasia, cancer, and/or an inflammatory complication to pouch surgery in an individual and assisting in the selection of therapy.

[0044] The term "individual," "subject," or "patient" typically includes humans, but also includes other animals such as, e.g., other primates, rodents, canines, felines, equines, ovines, porcines, and the like. [0045] As used herein, the term "substantially the same amino acid sequence" includes an amino acid sequence that is similar, but not identical to, the naturally-occurring amino acid sequence. For example, an amino acid sequence, i.e., polypeptide, that has substantially the same amino acid sequence as an 12 protein can have one or more modifications such as amino acid additions, deletions, or substitutions relative to the amino acid sequence of the naturally- occurring 12 protein, provided that the modified polypeptide retains substantially at least one biological activity of 12 such as immunoreactivity. Comparison for substantial similarity between amino acid sequences is usually performed with sequences between about 6 and 100 residues, preferably between about 10 and 100 residues, and more preferably between about 25 and 35 residues. A particularly useful modification of a polypeptide of the present invention, or a fragment thereof, is a modification that confers, for example, increased stability. Incorporation of one or more D-amino acids is a modification useful in increasing stability of a polypeptide or polypeptide fragment. Similarly, deletion or substitution of lysine residues can increase stability by protecting the polypeptide or polypeptide fragment against degradation.

[0046] The term "clinical factor" includes a symptom in an individual that is associated with pouch surgery. Examples of clinical factors include, without limitation, diarrhea, abdominal pain, cramping, fever, anemia, weight loss, anxiety, depression, and combinations thereof. In some embodiments, a prognosis of risk associated with pouch surgery (e.g., IPAA) is based upon a combination of analyzing a sample obtained from an individual to determine the presence, level, or genotype of one or more markers, determining whether the individual has one or more clinical factors, and optionally applying one or more statistical analyses.

[0047] In certain instances, the methods of the invention are used in order to prognosticate the progression of dysplasia, cancer, and/or an inflammatory complication after an individual has undergone pouch surgery. The methods can be used to monitor the disease state, both progression and regression. In certain embodiments, the results of a marker profile and/or statistical analysis are compared to those results obtained for the same individual at an earlier time. In some embodiments, the methods of the present invention can also be used to predict the progression of postoperative complications to pouch surgery, e.g., by determining a risk or likelihood for dysplasia, cancer, and/or an inflammatory complication to progress either rapidly or slowly in an individual based on the presence or level of at least one marker in a sample. In other embodiments, the methods of the present invention can also be used to predict the regression of postoperative complications to pouch surgery, e.g., by determining a risk or likelihood for dysplasia, cancer, and/or an inflammatory complication to regress either rapidly or slowly in an individual based on the presence or level of at least one marker in a sample.

[0048] In certain other instances, the methods of the invention are used in order to monitor drug efficacy in an individual receiving a drug useful for treating postoperative complications to pouch surgery. The methods of the present invention can be used to monitor drug efficacy by determining a marker profile, alone or in combination with the application of a statistical analysis, to determine the disease state (e.g. , presence or severity of complications to pouch surgery) of an individual after a therapeutic agent has been administered.

[0049] In some instances, the methods of the invention are used for optimizing therapy in an individual having one or more postoperative complications to pouch surgery such as, e.g., dysplasia, cancer, and/or an inflammatory complication. The methods of the invention can be used to optimize therapy by determining a marker profile, alone or in combination with the application of a statistical analysis, to determine the course of therapy for an individual before a therapeutic agent has been administered or to adjust the course of therapy for an individual after a therapeutic agent has been administered in order to optimize the therapeutic efficacy of the therapeutic agent. In certain instances, the results of a statistical analysis are compared to those results obtained for the same individual at an earlier time during the course of therapy. As such, a comparison of the results provides an indication for the need to change the course of therapy or an indication for the need to increase or decrease the dose of the current course of therapy.

[0050] The term "course of therapy" includes any therapeutic approach taken to relieve or prevent one or more symptoms (i.e., clinical factors) associated with pouch surgery. The term "course of therapy" encompasses administering any compound, drug, procedure, or regimen useful for improving the health of an individual with postoperative complications to pouch surgery and includes one or more therapeutic agents useful for treating cancer (e.g., when the postoperative complication is a dysplasia or cancer) or treating IBD (e.g., when the postoperative complication is inflammation of the gastrointestinal tract). One skilled in the art will appreciate that either the course of therapy or the dose of the current course of therapy can be changed, e.g., based upon the results obtained through applying a statistical analysis in accordance with the present invention. [0051] The term "therapeutically effective amount or dose" includes an amount or dose of a drug that is capable of achieving a therapeutic effect in an individual in need thereof. For example, a therapeutically effective amount or dose of a drug useful for treating postoperative complications to pouch surgery can be the amount or dose that is capable of preventing or relieving one or more symptoms associated with pouch surgery. The exact amount or dose can be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1 -3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding ( 1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0052] The term "gene" refers to the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region, such as the promoter and 3 '-untranslated region, respectively, as well as intervening sequences (introns) between individual coding segments (exons).

[0053] The term "genotype" refers to the genetic composition of an organism, including, for example, whether a diploid organism is heterozygous or homozygous for one or more variant alleles of interest.

[0054] The term "polymorphism" refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A "polymorphic site" refers to the locus at which divergence occurs. Preferred polymorphic sites have at least two alleles, each occurring at a particular frequency in a population. A polymorphic locus may be as small as one base pair {i.e., single nucleotide polymorphism or SNP). Polymorphic markers include restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu. The first identified allele is arbitrarily designated as the reference allele, and other alleles are designated as alternative alleles, "variant alleles," or "variances." The allele occurring most frequently in a selected population is sometimes referred to as the "wild-type" allele. Diploid organisms may be homozygous or heterozygous for the variant alleles. The variant allele may or may not produce an observable physical or biochemical characteristic ("phenotype") in an individual carrying the variant allele. For example, a variant allele may alter the enzymatic activity of a protein encoded by a gene of interest. [0055] The terms "miRNA," "microRNA" or "miR" are used interchangeably herein and include single-stranded RNA molecules of 21 -23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (non-coding RNA); instead each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA and finally into a functional miRNA. Mature miRs are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression.

Embodiments described herein include both diagnostic, prognostic, and therapeutic applications.

[0056] In quartile analysis, there are three numbers (values) that divide a range of data into four equal parts. The first quartile (also called the 'lower quartile') is the number below which lies the 25 percent of the bottom data. The second quartile (the 'median') divides the range in the middle and has 50 percent of the data below it. The third quartile (also called the 'upper quartile') has 75 percent of the data below it and the top 25 percent of the data above it. As a non-limiting example, quartile analysis can be applied to the concentration level of a marker such as an antibody or other protein marker described herein, such that a marker level in the first quartile (<25%) is assigned a value of 1 , a marker level in the second quartile (25- 50%) is assigned a value of 2, a marker level in the third quartile (51 %-<75%) is assigned a value of 3, and a marker level in the fourth quartile (75%- 100%) is assigned a value of 4.

[0057] As used herein, "quartile sum score" or "QSS" includes the sum of quartile scores for all of the markers of interest. As a non-limiting example, a quartile sum score for a panel of 6 markers (e.g., serological, protein, and/or genetic) may range from 6-24, wherein each of the individual markers is assigned a quartile score of 1 -4 based upon the presence or absence of the marker, the concentration level of the marker, or the genotype of the marker.

III. Description of the Embodiments

[0058] The present invention provides prognostic methods for predicting whether a patient will have a particular risk of developing postoperative complications such as dysplasia/cancer and/or one or more inflammatory complications (e.g., pouchitis) following a colectomy with the creation of an internal pouch. By identifying patients with a higher risk of developing postoperative complications to pouch surgery, the methods and systems described herein provide invaluable information to assess the presence and/or severity of such complications and treatment options. In particular embodiments, applying a statistical analysis to a profile of serological, protein, and/or genetic markers improves the accuracy of predicting a risk of developing dysplasia/cancer and/or an inflammatory complication such as pouchitis after pouch surgery, and also enables the selection of appropriate treatment options. Accordingly, with the present invention, it is possible to predict patients who will have a particular risk of postoperative complications to pouch surgery such as ΓΡΑΑ.

[0059] In one aspect, the present invention provides a method for predicting or determining a risk of developing dysplasia/cancer and/or an inflammatory complication such as pouchitis in an individual following a surgical procedure whereby the colon is removed and an internal pouch is created {e.g., IPAA), the method comprising:

(b) predicting or determining a risk {e.g., probability) that the individual will develop dysplasia/cancer and/or an inflammatory complication such as pouchitis following the surgical procedure based upon the marker profile.

[0060] In certain embodiments, the present invention provides a method for predicting or determining a risk of developing dysplasia/cancer and/or an inflammatory complication {e.g., pouchitis) in an individual following a surgical procedure whereby the colon is removed and an internal pouch is created {e.g., IPAA), the method comprising:

(b) comparing the level or genotype of each of the markers to a reference level or genotype to predict or determine a risk {e.g., probability) that the individual will develop dysplasia/cancer and/or an inflammatory complication {e.g., pouchitis) following the surgical procedure.

[0061] In other embodiments, the present invention provides a method for predicting or determining a risk of developing dysplasia/cancer and/or an inflammatory complication {e.g., pouchitis) in an individual following a surgical procedure whereby the colon is removed and an internal pouch is created {e.g., IPAA), the method comprising:

(b) applying a statistical analysis {e.g., quartile analysis, median analysis, etc.) to the marker profile to predict or determine a risk {e.g., probability) that the individual will develop dysplasia/cancer and/or an inflammatory complication (e.g., pouchitis) following the surgical procedure.

[0062] The presence, level, and/or genotype of one or more markers (e.g., marker profile) may be determined in a sample obtained from an individual prior to the surgical procedure (e.g., preoperatively) and/or following the surgical procedure (e.g., postoperatively). In particular embodiments, the presence, level, and/or genotype of one or more markers of interest is determined prior to the surgical procedure, e.g., at the time of colectomy. In some embodiments, the surgical procedure whereby the colon is removed and an internal pouch is created comprises an ileal pouch-anal anastomosis (ΓΡΑΑ) procedure. In other embodiments, the inflammatory complication is pouchitis (e.g., acute pouchitis, chronic pouchitis, active pouchitis, refractory pouchitis, and the like), Crohn's disease (CD) of the pouch, cuffitis (e.g., inflammation in the rectal muscular cuff), pouch stricture, pouch sinus, proximal small bowel bacterial overgrowth, irritable pouch syndrome (IPS), anismus, or combinations thereof.

[0063] In some embodiments, the marker is a serological marker selected from an anti- neutrophil antibody, an anti-Saccharomyces cerevisiae antibody, an antimicrobial antibody, an acute phase protein, an apolipoprotein, a defensin, a growth factor, a cytokine, a cadherin, a cellular adhesion molecule, and a combination thereof. In one embodiment, the anti- neutrophil antibody comprises an anti-neutrophil cytoplasmic antibody (ANCA) such as ANCA detected by an immunoassay (e.g., ELISA), a perinuclear anti-neutrophil cytoplasmic antibody (pANCA) such as pANCA detected by an immunohistochemical assay (e.g., IFA) or a DNAse-sensitive immunohistochemical assay, or a combination thereof. In another embodiment, the anti-Saccharomyces cerevisiae antibody comprises an anti-Saccharomyces cerevisiae immunoglobulin A (ASCA-IgA), anti-Saccharomyces cerevisiae immunoglobulin G (ASCA-IgG), or a combination thereof.

[0064] In yet another embodiment, the antimicrobial antibody comprises an anti-outer membrane protein C (anti-OmpC) antibody, an anti-I2 antibody, an anti-flagellin antibody, or a combination thereof. In certain instances, the anti-flagellin antibody comprises an anti- Cbir-1 flagellin antibody, an anti-flagellin X antibody, an anti-flagellin A antibody, an anti- flagellin B antibody, or a combination thereof. In a further embodiment, the acute phase protein is C-Reactive protein (CRP). In another embodiment, the apolipoprotein is serum amyloid A (SAA). In yet another embodiment, the defensin is β defensin (e.g., β defensin-1 (BD1 ) and/or β defensin-2 (BD2)). In still yet another embodiment, the growth factor is epidermal growth factor (EGF). In a further embodiment, the cytokine comprises TNF-a, IL- 6, IL-8, IL-12, IL-17, IL-23, or a combination thereof. In an additional embodiment, the cadherin is E-cadherin. In another embodiment, the cellular adhesion molecule comprises ICAM- 1 , VCAM- 1 , or a combination thereof.

[0065] In particular embodiments, the marker comprises or consists of at least one, two, three, four, five, six, or all seven of the following serological markers: ASCA-IgA, ASCA- IgG, anti-OmpC antibody, anti-CBir- 1 antibody, anti-I2 antibody, ANCA, and/or pANCA (e.g., pANCA IFA and/or DNAse-sensitive pANCA IF A).

[0066] In some instances, the marker profile comprises or consists of ANCA and ASCA IgA. In other instances, the marker profile comprises or consists of ANCA and ASCA IgG. In yet other instances, the marker profile comprises or consists of ANCA and anti-CBirl antibody. In still yet other instances, the marker profile comprises or consists of ANCA and anti-OmpC antibody. In further instances, the marker profile comprises or consists of ANCA and anti-I2 antibody. In yet further instances, the marker profile comprises or consists of ANCA and pANCA.

[0067] In some instances, the marker profile comprises or consists of ASCA IgA and ASCA IgG. In other instances, the marker profile comprises or consists of ASCA IgA and anti-CBirl antibody. In yet other instances, the marker profile comprises or consists of ASCA IgA and anti-OmpC antibody. In still yet other instances, the marker profile comprises or consists of ASCA IgA and anti-I2 antibody. In further instances, the marker profile comprises or consists of ASCA IgA and pANCA.

[0068] In some instances, the marker profile comprises or consists of ASCA IgG and anti- CBirl antibody. In other instances, the marker profile comprises or consists of ASCA IgG and anti-OmpC antibody. In yet other instances, the marker profile comprises or consists of ASCA IgG and anti-I2 antibody. In still yet other instances, the marker profile comprises or consists of ASCA IgG and pANCA.

[0069] In some instances, the marker profile comprises or consists of anti-CBirl antibody and anti-OmpC antibody. In other instances, the marker profile comprises or consists of anti- CBirl antibody and anti-I2 antibody. In yet other instances, the marker profile comprises or consists of anti-CBirl antibody and pANCA. In still yet other instances, the marker profile comprises or consists of anti-OmpC antibody and anti-I2 antibody. In further instances, the marker profile comprises or consists of anti-OmpC antibody and pANCA. In yet further instances, the marker profile comprises or consists of anti-I2 antibody and pANCA.

[0070] In some instances, the marker profile comprises or consists of ANCA, ASCA IgA, and ASCA IgG. In other instances, the marker profile comprises or consists of ANCA, ASCA IgA, and anti-CBirl antibody. In yet other instances, the marker profile comprises or consists of ANCA, ASCA IgA, and anti-OmpC antibody. In still yet other instances, the marker profile comprises or consists of ANCA, ASCA IgA, and anti-I2 antibody. In further instances, the marker profile comprises or consists of ANCA, ASCA IgA, and pANCA.

[0071] In some instances, the marker profile comprises or consists of ANCA, ASCA IgG, and anti-CBirl antibody. In other instances, the marker profile comprises or consists of ANCA, ASCA IgG, and anti-OmpC antibody. In yet other instances, the marker profile comprises or consists of ANCA, ASCA IgG, and anti-I2 antibody. In still yet other instances, the marker profile comprises or consists of ANCA, ASCA IgG, and pANCA.

[0072] In some instances, the marker profile comprises or consists of ANCA, anti-CBirl antibody, and anti-OmpC antibody. In other instances, the marker profile comprises or consists of ANCA, anti-CBirl antibody, and anti-I2 antibody. In yet other instances, the marker profile comprises or consists of ANCA, anti-CBirl antibody, and pANCA. In still yet other instances, the marker profile comprises or consists of ANCA, anti-OmpC antibody, and anti-I2 antibody. In further instances, the marker profile comprises or consists of ANCA, anti-OmpC antibody, and pANCA. In yet further instances, the marker profile comprises or consists of ANCA, anti-I2 antibody, and pANCA.

[0073] In some instances, the marker profile comprises or consists of ANCA, ASCA IgA, ASCA IgG, and anti-CBirl antibody. In other instances, the marker profile comprises or consists of ANCA, ASCA IgA, ASCA IgG, and anti-OmpC antibody. In yet other instances, the marker profile comprises or consists of ANCA, ASCA IgA, ASCA IgG, and anti-I2 antibody. In still yet other instances, the marker profile comprises or consists of ANCA, ASCA IgA, ASCA IgG, and pANCA.

[0074] In some instances, the marker profile comprises or consists of ANCA, ASCA IgG, anti-CBirl antibody, and anti-OmpC antibody. In other instances, the marker profile comprises or consists of ANCA, ASCA IgG, anti-CBirl antibody, and anti-I2 antibody. In yet other instances, the marker profile comprises or consists of ANCA, ASCA IgG, anti- CBirl antibody, and pANCA. In still yet other instances, the marker profile comprises or consists of ANCA, anti-CBirl antibody, anti-OmpC antibody, and anti-I2 antibody. In further instances, the marker profile comprises or consists of ANCA, anti-CBirl antibody, anti-OmpC antibody, and pANCA. In yet further instances, the marker profile comprises or consists of ANCA, anti-OmpC antibody, anti-I2 antibody, and pANCA.

[0075] In some instances, the marker profile comprises or consists of ANCA, ASCA IgA, ASCA IgG, anti-CBirl antibody, and anti-OmpC antibody. In other instances, the marker profile comprises or consists of ANCA, ASCA IgA, ASCA IgG, anti-CBirl antibody, and anti-I2 antibody. In yet other instances, the marker profile comprises or consists of ANCA, ASCA IgA, ASCA IgG, anti-CBirl antibody, and pANCA.

[0076] In some instances, the marker profile comprises or consists of ANCA, ASCA IgA, ASCA IgG, anti-CBirl antibody, anti-OmpC antibody, and anti-I2 antibody. In other instances, the marker profile comprises or consists of ANCA, ASCA IgA, ASCA IgG, anti- CBirl antibody, anti-OmpC antibody, and pANCA. In yet other instances, the marker profile comprises or consists of ANCA, ASCA IgA, ASCA IgG, anti-CBirl antibody, anti-OmpC antibody, anti-I2 antibody, and pANCA.

[0077] In certain instances, at least one, two, three, four, five, six, seven, eight, nine, ten, or more of the following markers may be included in the marker profiles of the invention: C- reactive protein (CRP); serum amyloid A (SAA); β defensin-1 (BD1 ); β defensin-2 (BD2); epidermal growth factor (EGF); TNF-a; IL-6; IL-8; IL-12; IL-17; IL-23; E-cadherin; ICAM- 1 ; VCAM-1 ; NOD2; and combinations thereof.

[0078] The presence or (concentration) level of the serological marker can be detected (e.g., determined, measured, analyzed, etc.) with a hybridization assay, amplification-based assay, immunoassay, immunohistochemical assay, or a combination thereof. Non-limiting examples of assays, techniques, and kits for detecting or determining the presence or level of one or more serological markers in a sample are described in Section VI below.

[0079] In other embodiments, the marker is a genetic marker selected from at least one of the genes set forth in Tables 1 A-1 E (e.g., Table 1 A, IB, 1 C, ID, and/or IE). In particular embodiments, the genetic marker is NOD2. The genotype of the genetic marker can be detected (e.g., determined, analyzed, etc.) by genotyping an individual for the presence or absence of one or more variant alleles such as, for example, one or more single nucleotide polymorphisms (SNPs) in one or more genetic markers. In some embodiments, the SNP is at least one of the SNPs set forth in Tables I B-IE (e.g., Table IB, 1 C, ID, and/or IE). Non- limiting examples of techniques for detecting or determining the genotype of one or more genetic markers in a sample are described in Section VII below. In certain embodiments, the genetic marker is NOD2 and the SNP is SNP8 (R702W), SNP12 (G908R), and/or SNP13 (1007fs). In certain instances, the presence or absence of one or more NOD2 SNPs is determined in combination with the presence or level of at least one, two, three, four, five, six, or all seven of the following serological markers: ASCA-IgA, ASCA-IgG, anti-OmpC antibody, anti-CBir-1 antibody, anti-I2 antibody, ANCA, and/or pANCA (e.g., pANCA IFA and/or DNAse-sensitive pANCA IFA).

[0080] In the methods of the present invention, the marker profile can be determined by detecting the presence, level, or genotype of at least one, two, three, four, five, six, seven, eight, nine, or ten markers. In particular embodiments, the sample is serum, plasma, whole blood, and/or stool. In other embodiments, the individual is diagnosed with ulcerative colitis (UC), indeterminate colitis (IC), or familial polyposis preoperatively and is in need of surgery such as pouch surgery.

[0081] In certain embodiments, the statistical analysis applied to the marker profile can comprise any of a variety of statistical methods, models, and algorithms described in Section IX below. In some embodiments, the statistical analysis is a median analysis. For median analysis, the presence and/or level of a single marker or a combination of markers may be classified as either below median, e.g., lower than a reference value such as, e.g., a median concentration level, or above median, e.g., higher than a reference value such as, e.g., a median concentration level. In particular embodiments, the statistical analysis is a quartile analysis. In some instances, the quartile analysis converts the presence, level, or genotype of a single marker into a quartile score. As a non-limiting example, the marker profile can correspond to a quartile sum score (QSS) for the individual that is obtained by summing the quartile score for a combination of markers. In certain embodiments, the pANCA marker is a binary rather than a numerical variable since its value is either positive or negative. In some instances, the quartile scoring for pANCA may be inverted, such that a positive status is scored as "1" and a negative status is scored as "4".

[0082] In particular embodiments, the methods described herein provide a prediction of (at least) about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range therein) probability or likelihood that one or more complications from pouch surgery would occur by a specific month or year after the surgery (e.g., Month 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 ; Year 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, etc.) based on an individual's marker profile, e.g., the individual 's quartile score for one marker or QSS for multiple markers, optionally in combination with the presence or absence of one or more variant alleles in one or more genetic markers, e.g., NOD2.

[0083] In certain embodiments, an individual is predicted to have a high or increased risk, probability, or likelihood of developing dysplasia or cancer when anti-OmpC antibody levels in a sample such as a serum sample are below median using median analysis and/or below median (e.g., Q2) using quartile analysis (see, Tables 6A and 6B in Example 8). In other embodiments, an individual is predicted to have a high or increased risk, probability, or likelihood of developing CD of the pouch when ANCA, anti-CBir-1 antibody, anti-OmpC antibody, and/or pANCA levels are above median using median analysis and/or above median (e.g., Q3 and/or Q4) using quartile analysis (see, Tables 7A and 7B in Example 8). In further embodiments, an individual is predicted to have a high or increased risk, probability, or likelihood of developing surgical complications when ANCA levels are below median using median analysis and/or below median (e.g., Ql and/or Q2) using quartile analysis (see, Tables 8 A and 8B in Example 8). In yet further embodiments, an individual is predicted to have a high or increased risk, probability, or likelihood of developing CD complications or a particular CD phenotype such as a fibrostenotic subtype of CD or a fistulating subtype of CD when anti-CBir-1 antibody and/or anti-OmpC antibody levels are above median using median analysis and/or above median (e.g., Q3 and/or Q4) using quartile analysis (see, Tables 9A and 9B in Example 8).

[0084] In some embodiments, the methods of the present invention can further comprise recommending a course of therapy for the individual based upon the marker profile. In other embodiments, the methods of the present invention can further comprise sending the results of the prediction to a clinician.

IV. Clinical Subtypes of IBD

[0085] Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the large intestine and small intestine. The main forms of IBD are Crohn's disease (CD) and ulcerative colitis (UC). Other less common forms of IBD include, e.g., indeterminate colitis (IC), collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome, infective colitis, and the like. U.S. Patent Publication 200801 3 1439, entitled "Methods of Diagnosing Inflammatory Bowel Disease" is incorporated herein by reference for all purposes.

A. Crohn's Disease

[0086] Crohn's disease (CD) is a disease of chronic inflammation that can involve any part of the gastrointestinal tract. Commonly, the distal portion of the small intestine, i.e., the ileum, and the cecum are affected. In other cases, the disease is confined to the small intestine, colon, or anorectal region. CD occasionally involves the duodenum and stomach, and more rarely the esophagus and oral cavity.

[0087] The variable clinical manifestations of CD are, in part, a result of the varying anatomic localization of the disease. The most frequent symptoms of CD are abdominal pain, diarrhea, and recurrent fever. CD is commonly associated with intestinal obstruction or fistula, an abnormal passage between diseased loops of bowel. CD also includes

complications such as inflammation of the eye, joints, and skin, liver disease, kidney stones, and amyloidosis. In addition, CD is associated with an increased risk of intestinal cancer.

[0088] Several features are characteristic of the pathology of CD. The inflammation associated with CD, known as transmural inflammation, involves all layers of the bowel wall. Thickening and edema, for example, typically also appear throughout the bowel wall, with fibrosis present in long-standing forms of the disease. The inflammation characteristic of CD is discontinuous in that segments of inflamed tissue, known as "skip lesions," are separated by apparently normal intestine. Furthermore, linear ulcerations, edema, and inflammation of the intervening tissue lead to a "cobblestone" appearance of the intestinal mucosa, which is distinctive of CD.

[0089] A hallmark of CD is the presence of discrete aggregations of inflammatory cells, known as granulomas, which are generally found in the submucosa. Some CD cases display typical discrete granulomas, while others show a diffuse granulomatous reaction or a nonspecific transmural inflammation. As a result, the presence of discrete granulomas is indicative of CD, although the absence of granulomas is also consistent with the disease. Thus, transmural or discontinuous inflammation, rather than the presence of granulomas, is a preferred diagnostic indicator of CD (Rubin and Farber, Pathology (Second Edition), Philadelphia, J.B. Lippincott Company (1994)). [0090] Crohn's disease may be categorized by the behavior of disease as it progresses. This was formalized in the Vienna classification of Crohn's disease. See, Gasche et ai , Inflamm. Bowel Dis. , 6:8- 15 (2000). There are three categories of disease presentation in Crohn's disease: ( 1 ) stricturing, (2) penetrating, and (3) inflammatory. Stricturing disease causes narrowing of the bowel which may lead to bowel obstruction or changes in the caliber of the feces. Penetrating disease creates abnormal passageways (fistulae) between the bowel and other structures such as the skin. Inflammatory disease (also known as non-stricturing, non-penetrating disease) causes inflammation without causing strictures or fistulae.

[0091] As such, Crohn's disease represents a number of heterogeneous disease subtypes that affect the gastrointestinal tract and may produce similar symptoms. As used herein in reference to CD, the term "clinical subtype" includes a classification of CD defined by a set of clinical criteria that distinguish one classification of CD from another. As non-limiting examples, subjects with CD can be classified as having stricturing {e.g., internal stricturing), penetrating {e.g., internal penetrating), or inflammatory disease as described herein, or these subjects can additionally or alternatively be classified as having fibrostenotic disease, small bowel disease, internal perforating disease, perianal fistulizing disease, UC-like disease, the need for small bowel surgery, the absence of features of UC, or combinations thereof.

[0092] In certain instances, subjects with CD can be classified as having complicated CD, which is a clinical subtype characterized by stricturing or penetrating phenotypes. In certain other instances, subjects with CD can be classified as having a form of CD characterized by one or more of the following complications: fibrostenosis, internal perforating disease, and the need for small bowel surgery. In further instances, subjects with CD can be classified as having an aggressive form of fibrostenotic disease requiring small bowel surgery. Criteria relating to these subtypes have been described, for example, in Gasche et ai, Inflamm. Bowel Dis. , 6:8- 15 (2000); Abreu et ai , Gastroenterology, 123:679-688 (2002); Vasiliauskas et al , Gut, 47:487-496 (2000); Vasiliauskas et ai, Gastroenterology, 1 10: 1810-1819 (1996); and Greenstein et ai, Gut, 29:588-592 (1988).

[0093] The "fibrostenotic subtype" of CD is a classification of CD characterized by one or more accepted characteristics of fibrostenosing disease. Such characteristics of

fibrostenosing disease include, but are not limited to, documented persistent intestinal obstruction or an intestinal resection for an intestinal obstruction. The fibrostenotic subtype of CD can be accompanied by other symptoms such as perforations, abscesses, or fistulae, and can further be characterized by persistent symptoms of intestinal blockage such as nausea, vomiting, abdominal distention, and inability to eat solid food. Intestinal X-rays of patients with the fibrostenotic subtype of CD can show, for example, distention of the bowel before the point of blockage.

[0094] The requirement for small bowel surgery in a subject with the fibrostenotic subtype of CD can indicate a more aggressive form of this subtype. Additional subtypes of CD are also known in the art and can be identified using defined clinical criteria. For example, internal perforating disease is a clinical subtype of CD defined by current or previous evidence of entero-enteric or entero- vesicular fistulae, intra-abdominal abscesses, or small bowel perforation. Perianal perforating disease is a clinical subtype of CD defined by current or previous evidence of either perianal fistulae or abscesses or rectovaginal fistula. The UC- like clinical subtype of CD can be defined by current or previous evidence of left-sided colonic involvement, symptoms of bleeding or urgency, and crypt abscesses on colonic biopsies. Disease location can be classified based on one or more endoscopic, radiologic, or pathologic studies.

[0095] One skilled in the art understands that overlap can exist between clinical subtypes of CD and that a subject having CD can have more than one clinical subtype of CD. For example, a subject having CD can have the fibrostenotic subtype of CD and can also meet clinical criteria for a clinical subtype characterized by the need for small bowel surgery or the internal perforating disease subtype. Similarly, the markers described herein can be associated with more than one clinical subtype of CD.

B. Ulcerative Colitis

[0096] Ulcerative colitis (UC) is a disease of the large intestine characterized by chronic diarrhea with cramping, abdominal pain, rectal bleeding, loose discharges of blood, pus, and mucus. The manifestations of UC vary widely. A pattern of exacerbations and remissions typifies the clinical course for about 70% of UC patients, although continuous symptoms without remission are present in some patients with UC. Local and systemic complications of UC include arthritis, eye inflammation such as uveitis, skin ulcers, and liver disease. In addition, UC, and especially the long-standing, extensive form of the disease is associated with an increased risk of colon carcinoma.

[0097] UC is a diffuse disease that usually extends from the most distal part of the rectum for a variable distance proximally. The term "left-sided colitis" describes an inflammation that involves the distal portion of the colon, extending as far as the splenic flexure. Sparing of the rectum or involvement of the right side (proximal portion) of the colon alone is unusual in UC. The inflammatory process of UC is limited to the colon and does not involve, for example, the small intestine, stomach, or esophagus. In addition, UC is distinguished by a superficial inflammation of the mucosa that generally spares the deeper layers of the bowel wall. Crypt abscesses, in which degenerated intestinal crypts are filled with neutrophils, are also typical of UC (Rubin and Farber, supra).

[0098] In certain instances, with respect to UC, the variability of symptoms reflect differences in the extent of disease (i.e., the amount of the colon and rectum that are inflamed) and the intensity of inflammation. Disease starts at the rectum and moves "up" the colon to involve more of the organ. UC can be categorized by the amount of colon involved. Typically, patients with inflammation confined to the rectum and a short segment of the colon adjacent to the rectum have milder symptoms and a better prognosis than patients with more widespread inflammation of the colon.

[0099] In comparison with CD, which is a patchy disease with frequent sparing of the rectum, UC is characterized by a continuous inflammation of the colon that usually is more severe distally than proximally. The inflammation in UC is superficial in that it is usually limited to the mucosal layer and is characterized by an acute inflammatory infiltrate with neutrophils and crypt abscesses. In contrast, CD affects the entire thickness of the bowel wall with granulomas often, although not always, present. Disease that terminates at the ileocecal valve, or in the colon distal to it, is indicative of UC, while involvement of the terminal ileum, a cobblestone-like appearance, discrete ulcers, or fistulas suggests CD.

[0100] The different types of ulcerative colitis are classified according to the location and the extent of inflammation. As used herein in reference to UC, the term "clinical subtype" includes a classification of UC defined by a set of clinical criteria that distinguish one classification of UC from another. As non-limiting examples, subjects with UC can be classified as having ulcerative proctitis, proctosigmoiditis, left-sided colitis, pancolitis, fulminant colitis, and combinations thereof. Criteria relating to these subtypes have been described, for example, in Kornbluth et ah , Am. J. Gastroenterol., 99: 1371 -85 (2004).

[0101] Ulcerative proctitis is a clinical subtype of UC defined by inflammation that is limited to the rectum. Proctosigmoiditis is a clinical subtype of UC which affects the rectum and the sigmoid colon. Left-sided colitis is a clinical subtype of UC which affects the entire left side of the colon, from the rectum to the place where the colon bends near the spleen and begins to run across the upper abdomen (the splenic flexure). Pancolitis is a clinical subtype of UC which affects the entire colon. Fulminant colitis is a rare, but severe form of pancolitis. Patients with fulminant colitis are extremely ill with dehydration, severe abdominal pain, protracted diarrhea with bleeding, and even shock.

[0102] In some embodiments, classification of the clinical subtype of UC is important in planning an effective course of treatment. While ulcerative proctitis, proctosigmoiditis, and left-sided colitis can be treated with local agents introduced through the anus, including steroid-based or other enemas and foams, pancolitis must be treated with oral medication so that active ingredients can reach all of the affected portions of the colon.

[0103] One skilled in the art understands that overlap can exist between clinical subtypes of UC and that a subject having UC can have more than one clinical subtype of UC. Similarly, the prognostic markers described herein can be associated with more than one clinical subtype of UC.

C. Indeterminate Colitis

[0104] Indeterminate colitis (IC) is a clinical subtype of IBD that includes both features of CD and UC. Such an overlap in the symptoms of both diseases can occur temporarily {e.g., in the early stages of the disease) or persistently (e.g., throughout the progression of the disease) in patients with IC. Clinically, IC is characterized by abdominal pain and diarrhea with or without rectal bleeding. For example, colitis with intermittent multiple ulcerations separated by normal mucosa is found in patients with the disease. Histologically, there is a pattern of severe ulceration with transmural inflammation. The rectum is typically free of the disease and the lymphoid inflammatory cells do not show aggregation. Although deep slitlike fissures are observed with foci of myocytolysis, the intervening mucosa is typically minimally congested with the preservation of goblet cells in patients with IC.

V. Prognostic Markers

[0105] A variety of prognostic markers, including biochemical markers, serological markers, protein markers, genetic markers, and other clinical or echographic characteristics, are suitable for use in the methods of the present invention. In certain aspects, the prognostic methods described herein utilize the application of an algorithm (e.g., statistical analysis) to the presence, concentration level, or genotype determined for one or more of the markers to aid or assist in providing a prediction of whether a patient will have a particular risk of developing postoperative complications such as dysplasia, cancer, and/or one or more inflammatory complications (e.g., pouchitis) following pouch surgery.

[0106] Non-limiting examples of suitable markers include: (i) biochemical, serological, and protein markers such as, e.g., anti-neutrophil antibodies, anti-Saccharomyces cerevisiae antibodies, antimicrobial antibodies, acute phase proteins, apolipoproteins, cytokines, growth factors, defensins, cadherins, cellular adhesion molecules, and combinations thereof; and (ii) genetic markers such as, e.g., any of the genes set forth in Tables 1 A- 1E (e.g., NOD2) and the miRNAs in Table 2.

A. Cytokines

[0107] The determination of the presence or level of at least one cytokine in a sample is particularly useful in the present invention. As used herein, the term "cytokine" includes any of a variety of polypeptides or proteins secreted by immune cells that regulate a range of immune system functions and encompasses small cytokines such as chemokines. The term "cytokine" also includes adipocytokines, which comprise a group of cytokines secreted by adipocytes that function, for example, in the regulation of body weight, hematopoiesis, angiogenesis, wound healing, insulin resistance, the immune response, and the inflammatory response.

[0108] In certain aspects, the presence or level of at least one cytokine including, but not limited to, TNF-a, TNF-related weak inducer of apoptosis (TWEAK), osteoprotegerin (OPG), IFN-a, IFN-β, IFN-γ, IL-l a, IL- Ι β, IL- 1 receptor antagonist (IL-lra), IL-2, IL-4, IL- 5, IL-6, soluble IL-6 receptor (sIL-6R), IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-23, and IL-27 is determined in a sample. In certain other aspects, the presence or level of at least one chemokine such as, for example, CXCLl/GROl/GROa, CXCL2/GR02, CXCL3/GR03, CXCL4/PF-4, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2,

CXCL9/MIG, CXCLl O/IP-10, CXCL1 1/I-TAC, CXCL12/SDF-1 , CXCL13/BCA-1 , CXCL14/BRAK, CXCL15, CXCL16, CXCL17/DMC, CCLl , CCL2/MCP-1 , CCL3/MIP-l a, CCL4/MIP-i p, CCL5/R ANTES, CCL6/C10, CCL7/MCP-3, CCL8/MCP-2, CCL9/CCL10, CCLl 1 /Eotaxin, CCL12/MCP-5, CCL13/MCP-4, CCL14/HCC-1 , CCL15/MIP-5,

CCL16/LEC, CCL17/TARC, CCL18/MIP-4, CCL19/MIP-3p, CCL20/MIP-3a, CCL21/SLC, CCL22/MDC, CCL23/MPIF1 , CCL24/Eotaxin-2, CCL25/TECK, CCL26/Eotaxin-3, CCL27/CTACK, CCL28/MEC, CL1 , CL2, and CX₃CL1 is determined in a sample. In certain further aspects, the presence or level of at least one adipocytokine including, but not limited to, leptin, adiponectin, resistin, active or total plasminogen activator inhibitor- 1 (PAI- 1 ), visfatin, and retinol binding protein 4 (RBP4) is determined in a sample. Preferably, the presence or level of IL-6, IL- Ι β, and/or TWEAK is determined.

[0109] In certain instances, the presence or level of a particular cytokine is detected at the level of mRNA expression with an assay such as, for example, a hybridization assay or an amplification-based assay. In certain other instances, the presence or level of a particular cytokine is detected at the level of protein expression using, for example, an immunoassay (e.g., ELISA) or an immunohistochemical assay. Suitable ELISA kits for determining the presence or level of a cytokine such as IL-6, IL-Ι β, or TWEAK in a serum, plasma, saliva, or urine sample are available from, e.g., R&D Systems, Inc. (Minneapolis, MN), Neogen Corp. (Lexington, KY), Alpco Diagnostics (Salem, NH), Assay Designs, Inc. (Ann Arbor, MI), BD Biosciences Pharmingen (San Diego, CA), Invitrogen (Camarillo, CA), Calbiochem (San Diego, CA), CHEMICON International, Inc. (Temecula, CA), Antigenix America Inc.

(Huntington Station, NY), QIAGEN Inc. (Valencia, CA), Bio-Rad Laboratories, Inc.

(Hercules, CA), and/or Bender MedSystems Inc. (Burlingame, CA).

[0110] The human IL-6 polypeptide sequence is set forth in, e.g. , Genbank Accession No. NP_000591 (SEQ ID NO: l ). The human IL-6 mRNA (coding) sequence is set forth in, e.g. , Genbank Accession No. NM_000600 (SEQ ID NO:2). One skilled in the art will appreciate that IL-6 is also known as interferon beta 2 (IFNB2), HGF, HSF, and BSF2.

[0111] The human IL-Ι β polypeptide sequence is set forth in, e.g., Genbank Accession No. NP_000567 (SEQ ID NO:3). The human IL- Ι β mRNA (coding) sequence is set forth in, e.g., Genbank Accession No. NM_000576 (SEQ ID NO:4). One skilled in the art will appreciate that ΓΕ- Ι β is also known as IL1F2 and IL-lbeta.

[0112] The human TWEAK polypeptide sequence is set forth in, e.g., Genbank Accession Nos. NP_003800 (SEQ ID NO:5) and AAC51923. The human TWEAK mRNA (coding) sequence is set forth in, e.g., Genbank Accession Nos. NM_003809 (SEQ ID NO:6) and BC 104420. One skilled in the art will appreciate that TWEAK is also known as tumor necrosis factor ligand superfamily member 12 (TNFSF12), AP03 ligand (AP03L), CD255, DR3 ligand, growth factor-inducible 14 (Fnl4) ligand, and UNQ181/PRO207. B. Growth Factors

[0113] The determination of the presence or level of one or more growth factors in a sample is also useful in the present invention. As used herein, the term "growth factor" includes any of a variety of peptides, polypeptides, or proteins that are capable of stimulating cellular proliferation and/or cellular differentiation.

[0114] In certain aspects, the presence or level of at least one growth factor including, but not limited to, epidermal growth factor (EGF), heparin-binding epidermal growth factor (HB- EGF), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF; also known as SERPINF1 ), amphiregulin (AREG; also known as schwannoma-derived growth factor (SDGF)), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), transforming growth factor-a (TGF-a), transforming growth factor-β (TGF-β), bone morphogenetic proteins (e.g., BMP 1 -BMP 15), platelet-derived growth factor (PDGF), nerve growth factor (NGF), β-nerve growth factor (β-NGF), neurotrophic factors (e.g., brain- derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4 (NT4), etc.), growth differentiation factor-9 (GDF-9), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), myostatin (GDF-8), erythropoietin (EPO), and thrombopoietin (TPO) is determined in a sample. Preferably, the presence or level of EGF is determined.

[0115] In certain instances, the presence or level of a particular growth factor is detected at the level of mRNA expression with an assay such as, for example, a hybridization assay or an amplification-based assay. In certain other instances, the presence or level of a particular growth factor is detected at the level of protein expression using, for example, an

immunoassay (e.g., ELISA) or an immunohistochemical assay. Suitable ELISA kits for determining the presence or level of a growth factor such as EGF in a serum, plasma, saliva, or urine sample are available from, e.g., Antigenix America Inc. (Huntington Station, NY), Promega (Madison, WI), R&D Systems, Inc. (Minneapolis, MN), Invitrogen (Camarillo, CA), CHEMICON International, Inc. (Temecula, CA), Neogen Corp. (Lexington, KY), PeproTech (Rocky Hill, NJ), Alpco Diagnostics (Salem, NH), Pierce Biotechnology, Inc. (Rockford, IL), and/or Abazyme (Needham, MA).

[0116] The human epidermal growth factor (EGF) polypeptide sequence is set forth in, e.g. , Genbank Accession No. NP_001954 (SEQ ID NO:7). The human EGF mRNA (coding) sequence is set forth in, e.g. , Genbank Accession No. NM_001963 (SEQ ID NO:8). One skilled in the art will appreciate that EGF is also known as beta-urogastrone, URG, and HOMG4.

C. Anti-Neutrophil Antibodies

[0117] The determination of ANCA levels and/or the presence or absence of pANCA in a sample is also useful in the present invention. As used herein, the term "anti-neutrophil cytoplasmic antibody" or "ANCA" includes antibodies directed to cytoplasmic and/or nuclear components of neutrophils. ANCA activity can be divided into several broad categories based upon the ANCA staining pattern in neutrophils: (1 ) cytoplasmic neutrophil staining without perinuclear highlighting (cANCA); (2) perinuclear staining around the outside edge of the nucleus (pANCA); (3) perinuclear staining around the inside edge of the nucleus (NSNA); and (4) diffuse staining with speckling across the entire neutrophil (SAPPA). In certain instances, pANCA staining is sensitive to DNase treatment. The term ANCA encompasses all varieties of anti-neutrophil reactivity, including, but not limited to, cANCA, pANCA, NSNA, and SAPPA. Similarly, the term ANCA encompasses all immunoglobulin isotypes including, without limitation, immunoglobulin A and G.

[0118] ANCA levels in a sample from an individual can be determined, for example, using an immunoassay such as an enzyme-linked immunosorbent assay (ELISA) with alcohol-fixed neutrophils {see, e.g., Example 1 ). The presence or absence of a particular category of ANCA such as pANCA can be determined, for example, using an immunohistochemical assay such as an indirect fluorescent antibody (IF A) assay. In certain embodiments, the presence or absence of pANCA in a sample is determined using an immunofluorescence assay with DNase-treated, fixed neutrophils {see, e.g., Example 2). In addition to fixed neutrophils, antibodies directed against human antibodies can be used for detection. Antigens specific for ANCA are also suitable for determining ANCA levels, including, without limitation, unpurified or partially purified neutrophil extracts; purified proteins, protein fragments, or synthetic peptides such as histone H I or ANCA-reactive fragments thereof {see, e.g., U.S. Patent No. 6,074,835); histone HI -like antigens, porin antigens, Bacteroides antigens, or ANCA-reactive fragments thereof {see, e.g., U.S. Patent No. 6,033,864);

secretory vesicle antigens or ANCA-reactive fragments thereof {see, e.g., U.S. Patent Application No. 08/804, 106); and anti-ANCA idiotypic antibodies. One skilled in the art will appreciate that the use of additional antigens specific for ANCA is within the scope of the present invention. D. Anti-Saccharomyces cerevisiae Antibodies

[0119] The determination of the presence or level of ASCA (e.g., ASCA-IgA, ASCA-lgG, ASCA-IgM, etc.) in a sample is also useful in the present invention. The term "anti- Saccharomyces cerevisiae immunoglobulin A" or "ASCA-IgA" includes antibodies of the immunoglobulin A isotype that react specifically with 5. cerevisiae. Similarly, the term "anti-Saccharomyces cerevisiae immunoglobulin G" or "ASCA-lgG" includes antibodies of the immunoglobulin G isotype that react specifically with S. cerevisiae.

[0120] The determination of whether a sample is positive for ASCA-IgA or ASCA-lgG is made using an antibody specific for human antibody sequences or an antigen specific for ASCA. Such an antigen can be any antigen or mixture of antigens that is bound specifically by ASCA-IgA and/or ASCA-lgG. Although ASCA antibodies were initially characterized by their ability to bind S. cerevisiae, those of skill in the art will understand that an antigen that is bound specifically by ASCA can be obtained from S. cerevisiae or from a variety of other sources so long as the antigen is capable of binding specifically to ASCA antibodies.

Accordingly, exemplary sources of an antigen specific for ASCA, which can be used to determine the levels of ASCA-IgA and/or ASCA-lgG in a sample, include, without limitation, whole killed yeast cells such as Saccharomyces or Candida cells; yeast cell wall mannan such as phosphopeptidomannan (PPM); oligosaccharides such as oligomannosides; neoglycolipids; anti-ASCA idiotypic antibodies; and the like. Different species and strains of yeast, such as S. cerevisiae strain Su l , Su2, CBS 1315, or BM 156, or Candida albicans strain VW32, are suitable for use as an antigen specific for ASCA-IgA and/or ASCA-lgG. Purified and synthetic antigens specific for ASCA are also suitable for use in determining the levels of ASCA-IgA and/or ASCA-lgG in a sample. Examples of purified antigens include, without limitation, purified oligosaccharide antigens such as oligomannosides. Examples of synthetic antigens include, without limitation, synthetic oligomannosides such as those described in U.S. Patent Publication No. 20030105060, e.g., D-Man β(1 -2) D-Man β(1-2) D- Man β( 1 -2) D-Man-OR, D-Man a(l -2) D-Man a( l-2) D-Man a(l-2) D-Man-OR, and D-Man a(l -3) D-Man <x(l -2) D-Man a(l -2) D-Man-OR, wherein R is a hydrogen atom, a Ci to C₂o alkyl, or an optionally labeled connector group.

[0121] Preparations of yeast cell wall mannans, e.g., PPM, can be used in determining the levels of ASCA-IgA and/or ASCA-lgG in a sample. Such water-soluble surface antigens can be prepared by any appropriate extraction technique known in the art, including, for example, by autoclaving, or can be obtained commercially (see, e.g., Lindberg et al , Gut, 33:909-913 (1992)). The acid-stable fraction of PPM is also useful in the statistical algorithms of the present invention (Sendid et al. , Clin. Diag. Lab. Immunol., 3:219-226 ( 1996)). An exemplary PPM that is useful in determining ASCA levels in a sample is derived from 5. uvarum strain ATCC #38926. Example 3 describes the preparation of yeast cell well mannan and an analysis of ASCA levels in a sample using an ELISA assay.

[0122] Purified oligosaccharide antigens such as oligomannosides can also be useful in determining the levels of ASCA-IgA and/or ASCA-IgG in a sample. The purified

oligomannoside antigens are preferably converted into neoglycolipids as described in, for example, Faille et al , Eur. J. Microbiol. Infect. Dis. , 1 1 :438-446 (1992). One skilled in the art understands that the reactivity of such an oligomannoside antigen with ASCA can be optimized by varying the mannosyl chain length (Frosh et al, Proc Natl. Acad. Sci. USA, 82: 1 194- 1 198 ( 1985)); the anomeric configuration (Fukazawa et al , In "Immunology of Fungal Disease," E. Kurstak (ed.), Marcel Dekker Inc., New York, pp. 37-62 (1989);

Nishikawa et al, Microbiol. Immunol., 34:825-840 (1990); Poulain et al, Eur. J. Clin.

Microbiol., 23:46-52 (1993); Shibata et al. , Arch. Biochem. Biophys., 243:338-348 (1985); Trinel et al, Infect. Immun. , 60:3845-3851 (1992)); or the position of the linkage (Kikuchi et al. , Planta, 190:525-535 (1993)).

[0123] Suitable oligomannosides for use in the methods of the present invention include, without limitation, an oligomannoside having the mannotetraose Man(l -3) Man( l -2) Man( l - 2) Man. Such an oligomannoside can be purified from PPM as described in, e.g., Faille et al, supra. An exemplary neoglycolipid specific for ASCA can be constructed by releasing the oligomannoside from its respective PPM and subsequently coupling the released

oligomannoside to 4-hexadecylaniline or the like.

E. Anti-Microbial Antibodies

[0124] The determination of the presence or level of anti-OmpC antibody in a sample is also useful in the present invention. As used herein, the term "anti-outer membrane protein C antibody" or "anti-OmpC antibody" includes antibodies directed to a bacterial outer membrane porin as described in, e.g., U.S. Patent No. 7, 138,237 and PCT Patent Publication No. WO 01/89361 . The term "outer membrane protein C" or "OmpC" refers to a bacterial porin that is immunoreactive with an anti-OmpC antibody. [0125] The level of anti-OmpC antibody present in a sample from an individual can be determined using an OmpC protein or a fragment thereof such as an immunoreactive fragment thereof. Suitable OmpC antigens useful in determining anti-OmpC antibody levels in a sample include, without limitation, an OmpC protein, an OmpC polypeptide having substantially the same amino acid sequence as the OmpC protein, or a fragment thereof such as an immunoreactive fragment thereof. As used herein, an OmpC polypeptide generally describes polypeptides having an amino acid sequence with greater than about 50% identity, preferably greater than about 60% identity, more preferably greater than about 70% identity, still more preferably greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with an OmpC protein, with the amino acid identity determined using a sequence alignment program such as CLUSTALW. Such antigens can be prepared, for example, by purification from enteric bacteria such as E. coli, by recombinant expression of a nucleic acid such as Genbank Accession No. K00541 , by synthetic means such as solution or solid phase peptide synthesis, or by using phage display. Example 4 describes the preparation of OmpC protein and an analysis of anti-OmpC antibody levels in a sample using an ELISA assay.

[0126] The determination of the presence or level of anti-I2 antibody in a sample is also useful in the present invention. As used herein, the term "anti-I2 antibody" includes antibodies directed to a microbial antigen sharing homology to bacterial transcriptional regulators as described in, e.g., U.S. Patent No. 6,309,643. The term "12" refers to a microbial antigen that is immunoreactive with an anti-I2 antibody. The microbial 12 protein is a polypeptide of 100 amino acids sharing some similarity weak homology with the predicted protein 4 from C. pasteurianum, Rv3557c from Mycobacterium tuberculosis, and a transcriptional regulator from Aquifex aeolicus. The nucleic acid and protein sequences for the 12 protein are described in, e.g., U.S. Patent No. 6,309,643.

[0127] The level of anti-I2 antibody present in a sample from an individual can be determined using an 12 protein or a fragment thereof such as an immunoreactive fragment thereof. Suitable 12 antigens useful in determining anti-I2 antibody levels in a sample include, without limitation, an 12 protein, an 12 polypeptide having substantially the same amino acid sequence as the 12 protein, or a fragment thereof such as an immunoreactive fragment thereof. Such 12 polypeptides exhibit greater sequence similarity to the 12 protein than to the C. pasteurianum protein 4 and include isotype variants and homologs thereof. As used herein, an 12 polypeptide generally describes polypeptides having an amino acid sequence with greater than about 50% identity, preferably greater than about 60% identity, more preferably greater than about 70% identity, still more preferably greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with a naturally-occurring 12 protein, with the amino acid identity determined using a sequence alignment program such as CLUSTALW. Such 12 antigens can be prepared, for example, by purification from microbes, by recombinant expression of a nucleic acid encoding an 12 antigen, by synthetic means such as solution or solid phase peptide synthesis, or by using phage display. Determination of anti-I2 antibody levels in a sample can be done by using an ELISA assay {see, e.g., Examples 5, 10, and 12) or a histological assay.

[0128] The determination of the presence or level of anti-flagellin antibody in a sample is also useful in the present invention. As used herein, the term "anti-flagellin antibody" includes antibodies directed to a protein component of bacterial flagella as described in, e.g., U.S. Patent No. 7,361 ,733 and PCT Patent Publication No. WO 03/053220. The term "flagellin" refers to a bacterial flagellum protein that is immunoreactive with an anti-flagellin antibody. Microbial flagellins are proteins found in bacterial flagellum that arrange themselves in a hollow cylinder to form the filament.

[0129] The level of anti-flagellin antibody present in a sample from an individual can be determined using a flagellin protein or a fragment thereof such as an immunoreactive fragment thereof. Suitable flagellin antigens useful in determining anti-flagellin antibody levels in a sample include, without limitation, a flagellin protein such as Cbir- 1 flagellin, flagellin X, flagellin A, flagellin B, fragments thereof, and combinations thereof, a flagellin polypeptide having substantially the same amino acid sequence as the flagellin protein, or a fragment thereof such as an immunoreactive fragment thereof. As used herein, a flagellin polypeptide generally describes polypeptides having an amino acid sequence with greater than about 50% identity, preferably greater than about 60% identity, more preferably greater than about 70% identity, still more preferably greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with a naturally-occurring flagellin protein, with the amino acid identity determined using a sequence alignment program such as CLUSTALW. Such flagellin antigens can be prepared, e.g., by purification from bacterium such as Helicobacter Bilis, Helicobacter mustelae, Helicobacter pylori, Butyrivibrio fibrisolvens, and bacterium found in the cecum, by recombinant expression of a nucleic acid encoding a flagellin antigen, by synthetic means such as solution or solid phase peptide synthesis, or by using phage display. Determination of anti-flagellin (e.g., anti-Cbir-1 ) antibody levels in a sample can be done by using an ELISA assay or a histological assay.

F. Acute Phase Proteins

[0130] The determination of the presence or level of one or more acute-phase proteins in a sample is also useful in the present invention. Acute-phase proteins are a class of proteins whose plasma concentrations increase (positive acute-phase proteins) or decrease (negative acute-phase proteins) in response to inflammation. This response is called the acute -phase reaction (also called acute-phase response). Examples of positive acute-phase proteins include, but are not limited to, C-reactive protein (CRP), D-dimer protein, mannose-binding protein, alpha 1 -antitrypsin, alpha 1 -antichymotrypsin, alpha 2-macroglobulin, fibrinogen, prothrombin, factor VIII, von Willebrand factor, plasminogen, complement factors, ferritin, serum amyloid P component, serum amyloid A (SAA), orosomucoid (alpha 1-acid glycoprotein, AGP), ceruloplasmin, haptoglobin, and combinations thereof. Non-limiting examples of negative acute-phase proteins include albumin, transferrin, transthyretin, transcortin, retinol-binding protein, and combinations thereof. Preferably, the presence or level of CRP and/or SAA is determined.

[0131] In certain instances, the presence or level of a particular acute-phase protein is detected at the level of mRNA expression with an assay such as, for example, a hybridization assay or an amplification-based assay. In certain other instances, the presence or level of a particular acute-phase protein is detected at the level of protein expression using, for example, an immunoassay (e.g., ELISA) or an immunohistochemical assay. For example, a sandwich colorimetric ELISA assay available from Alpco Diagnostics (Salem, NH) can be used to determine the level of CRP in a serum, plasma, urine, or stool sample. Similarly, an ELISA kit available from Biomeda Corporation (Foster City, CA) can be used to detect CRP levels in a sample. Other methods for determining CRP levels in a sample are described in, e.g., U.S. Patent Nos. 6,838,250 and 6,406,862; and U.S. Patent Publication Nos.

20060024682 and 20060019410. Additional methods for determining CRP levels include, e.g., immunoturbidimetry assays, rapid immunodiffusion assays, and visual agglutination assays.

[0132] C-reactive protein (CRP) is a protein found in the blood in response to inflammation (an acute-phase protein). CRP is typically produced by the liver and by fat cells (adipocytes). It is a member of the pentraxin family of proteins. The human CRP polypeptide sequence is set forth in, e.g., Genbank Accession No. NP_000558 (SEQ ID NO:9). The human CRP mRNA (coding) sequence is set forth in, e.g. , Genbank Accession No. NM_000567 (SEQ ID NO: 10). One skilled in the art will appreciate that CRP is also known as PTX l , MGC88244, and MGC149895.

G. Apolipoproteins

[0133] The determination of the presence or level of one or more apolipoproteins in a sample is also useful in the present invention. Apolipoproteins are proteins that bind to fats (lipids). They form lipoproteins, which transport dietary fats through the bloodstream.

Dietary fats are digested in the intestine and carried to the liver. Fats are also synthesized in the liver itself. Fats are stored in fat cells (adipocytes). Fats are metabolized as needed for energy in the skeletal muscle, heart, and other organs and are secreted in breast milk.

Apolipoproteins also serve as enzyme co-factors, receptor ligands, and lipid transfer carriers that regulate the metabolism of lipoproteins and their uptake in tissues. Examples of apolipoproteins include, but are not limited to, ApoA (e.g., ApoA-I, ApoA-II, ApoA-IV, ApoA-V), ApoB {e.g., ApoB48, ApoB l OO), ApoC {e.g., ApoC-I, ApoC-II, ApoC-III, ApoC- IV), ApoD, ApoE, ApoH, serum amyloid A (SAA), and combinations thereof. Preferably, the presence or level of SAA is determined.

[0134] In certain instances, the presence or level of a particular apolipoprotein is detected at the level of mRNA expression with an assay such as, for example, a hybridization assay or an amplification-based assay. In certain other instances, the presence or level of a particular apolipoprotein is detected at the level of protein expression using, for example, an immunoassay (e.g., ELISA) or an immunohistochemical assay. Suitable ELISA kits for determining the presence or level of SAA in a sample such as serum, plasma, saliva, urine, or stool are available from, e.g., Antigenix America Inc. (Huntington Station, NY), Abazyme (Needham, MA), USCN Life (Missouri City, TX), and/or U.S. Biological (Swampscott, MA).

[0135] Serum amyloid A (SAA) proteins are a family of apolipoproteins associated with high-density lipoprotein (HDL) in plasma. Different isoforms of SAA are expressed constitutively (constitutive SAAs) at different levels or in response to inflammatory stimuli (acute phase SAAs). These proteins are predominantly produced by the liver. The conservation of these proteins throughout invertebrates and vertebrates suggests SAAs play a highly essential role in all animals. Acute phase serum amyloid A proteins (A-SAAs) are secreted during the acute phase of inflammation. The human SAA polypeptide sequence is set forth in, e.g., Genbank Accession No. NP_000322 (SEQ ID NO: l 1 ). The human SAA mRNA (coding) sequence is set forth in, e.g. , Genbank Accession No. NM_000331 (SEQ ID NO: 12). One skilled in the art will appreciate that SAA is also known as PIG4, TP53I4, MGC 1 1 1216, and SAA 1 .

H. Defensins

[0136] The determination of the presence or level of one or more defensins in a sample is also useful in the present invention. Defensins are small cysteine-rich cationic proteins found in both vertebrates and invertebrates. They are active against bacteria, fungi, and many enveloped and nonenveloped viruses. They typically consist of 18-45 amino acids, including 6 (in vertebrates) to 8 conserved cysteine residues. Cells of the immune system contain these peptides to assist in killing phagocytized bacteria, for example, in neutrophil granulocytes and almost all epithelial cells. Most defensins function by binding to microbial cell membranes, and once embedded, forming pore-like membrane defects that allow efflux of essential ions and nutrients. Non-limiting examples of defensins include a-defensins (e.g., DEFAl , DEFAl A3, DEFA3, DEFA4), β-defensins (e.g., β defensin- l (DEFB l ), β defensin-2 (DEFB2), DEFB l 03 A/DEFB 103B to DEFB l 07 A/DEFB 107B, DEFB l 10 to DEFB l 33), and combinations thereof. Preferably, the presence or level of DEFB l and/or DEFB2 is determined.

[0137] In certain instances, the presence or level of a particular defensin is detected at the level of mRNA expression with an assay such as, for example, a hybridization assay or an amplification-based assay. In certain other instances, the presence or level of a particular defensin is detected at the level of protein expression using, for example, an immunoassay (e.g., ELISA) or an immunohistochemical assay. Suitable ELISA kits for determining the presence or level of DEFB l and/or DEFB2 in a sample such as serum, plasma, saliva, urine, or stool are available from, e.g., Alpco Diagnostics (Salem, NH), Antigenix America Inc. (Huntington Station, NY), PeproTech (Rocky Hill, NJ), and/or Alpha Diagnostic Intl. Inc. (San Antonio, TX).

[0138] β-defensins are antimicrobial peptides implicated in the resistance of epithelial surfaces to microbial colonization. They are the most widely distributed of all defensins, being secreted by leukocytes and epithelial cells of many kinds. For example, they can be found on the tongue, skin, cornea, salivary glands, kidneys, esophagus, and respiratory tract. The human DEFB 1 polypeptide sequence is set forth in, e.g. , Genbank Accession No.

NP_005209 (SEQ ID NO: 13). The human DEFB 1 mRNA (coding) sequence is set forth in, e.g., Genbank Accession No. NM_005218 (SEQ ID NO: 14). One skilled in the art will appreciate that DEFB 1 is also known as BD 1 , HBD 1 , DEFB-1 , DEFB 101 , and MGC51822. The human DEFB2 polypeptide sequence is set forth in, e.g. , Genbank Accession No.

NP_004933 (SEQ ID NO: 15). The human DEFB2 mRNA (coding) sequence is set forth in, e.g. , Genbank Accession No. NM_004942 (SEQ ID NO: 16). One skilled in the art will appreciate that DEFB2 is also known as SAP 1 , HBD-2, DEFB-2, DEFB 102, and DEFB4.

I. Cadherins

[0139] The determination of the presence or level of one or more cadherins in a sample is also useful in the present invention. Cadherins are a class of type- 1 transmembrane proteins which play important roles in cell adhesion, ensuring that cells within tissues are bound together. They are dependent on calcium (Ca²⁺) ions to function. The cadherin superfamily includes cadherins, protocadherins, desmogleins, and desmocollins, and more. In structure, they share cadherin repeats, which are the extracellular Ca²⁺-binding domains. Cadherins suitable for use in the present invention include, but are not limited to, CDH1 - E-cadherin (epithelial), CDH2 - N-cadherin (neural), CDH12 - cadherin 12, type 2 (N-cadherin 2), CDH3 - P-cadherin (placental),CDH4 - R-cadherin (retinal), CDH5 - VE-cadherin (vascular endothelial),CDH6 - K-cadherin (kidney), CDH7 - cadherin 7, type 2, CDH8 - cadherin 8, type 2, CDH9 - cadherin 9, type 2 (Tl -cadherin), CDHI O - cadherin 10, type 2 (T2-cadherin), CDH1 1 - OB-cadherin (osteoblast), CDH13 - T-cadherin - H-cadherin (heart), CDH15 - M- cadherin (myotubule), CDH16 - KSP-cadherin, CDH17 - LI cadherin (liver-intestine), CDH18 - cadherin 1 8, type 2, CDH19 - cadherin 19, type 2, CDH20 - cadherin 20, type 2, and CDH23 - cadherin 23, (neurosensory epithelium). Preferably, the presence or level of E- cadherin is determined.

[0140] In certain instances, the presence or level of a particular cadherin is detected at the level of mRNA expression with an assay such as, for example, a hybridization assay or an amplification-based assay. In certain other instances, the presence or level of a particular cadherin is detected at the level of protein expression using, for example, an immunoassay (e.g., ELISA) or an immunohistochemical assay. Suitable ELISA kits for determining the presence or level of E-cadherin in a sample such as serum, plasma, saliva, urine, or stool are available from, e.g., R&D Systems, Inc. (Minneapolis, MN) and/or GenWay Biotech, Inc. (San Diego, CA).

[0141] E-cadherin is a classical cadherin from the cadherin superfamily. It is a calcium dependent cell-cell adhesion glycoprotein comprised of five extracellular cadherin repeats, a transmembrane region, and a highly conserved cytoplasmic tail. The ectodomain of E- cadherin mediates bacterial adhesion to mammalian cells and the cytoplasmic domain is required for internalization. The human E-cadherin polypeptide sequence is set forth in, e.g., Genbank Accession No. NP_004351 (SEQ ID NO: 17). The human E-cadherin mRNA (coding) sequence is set forth in, e.g. , Genbank Accession No. NM_004360 (SEQ ID

NO: 18). One skilled in the art will appreciate that E-cadherin is also known as UVO, CDHE, ECAD, LCAM, Arc- 1 , CD324, and CDH1 .

J. Cellular Adhesion Molecules (IgSF CAMs)

[0142] The determination of the presence or level of one or more immunoglobulin superfamily cellular adhesion molecules in a sample is also useful in the present invention. As used herein, the term "immunoglobulin superfamily cellular adhesion molecule" (IgSF CAM) includes any of a variety of polypeptides or proteins located on the surface of a cell that have one or more immunoglobulin-like fold domains, and which function in intercellular adhesion and/or signal transduction. In many cases, IgSF CAMs are transmembrane proteins. Non-limiting examples of IgSF CAMs include Neural Cell Adhesion Molecules (NCAMs; e.g. , NCAM- 120, NCAM- 125, NCAM-140, NCAM-145, NCAM-180, NCAM- 185, etc.), Intercellular Adhesion Molecules (ICAMs, e.g., ICAM-1 , ICAM-2, ICAM-3, ICAM-4, and ICAM-5), Vascular Cell Adhesion Molecule-1 (VCAM-1 ), Platelet-Endothelial Cell

Adhesion Molecule- 1 (PECAM-1 ), LI Cell Adhesion Molecule (LI CAM), cell adhesion molecule with homology to LI CAM (close homolog of LI ) (CHL1), sialic acid binding Ig- like lectins (SIGLECs; e.g., SIGLEC-1 , SIGLEC-2, SIGLEC-3, SIGLEC-4, etc.), Nectins {e.g. , Nectin-1 , Nectin-2, Nectin-3, etc.), and Nectin-like molecules {e.g. , Necl-1 , Necl-2, Necl-3, Necl-4, and Necl-5). Preferably, the presence or level of ICAM-1 and/or VCAM-1 is determined.

1. Intercellular Adhesion Molecule-1 (ICAM-1)

[0143] ICAM- 1 is a transmembrane cellular adhesion protein that is continuously present in low concentrations in the membranes of leukocytes and endothelial cells. Upon cytokine stimulation, the concentrations greatly increase. ICAM-1 can be induced by IL-1 and TNFa and is expressed by the vascular endothelium, macrophages, and lymphocytes. In IBD, proinflammatory cytokines cause inflammation by upregulating expression of adhesion molecules such as ICAM- 1 and VCAM- 1 . The increased expression of adhesion molecules recruit more lymphocytes to the infected tissue, resulting in tissue inflammation {see, Goke et al, J., Gastroenterol , 32:480 ( 1997); and Rijcken et al, Gut, 51 :529 (2002)). ICAM-1 is encoded by the intercellular adhesion molecule 1 gene (ICAM 1 ; Entrez GeneID:3383 ;

Genbank Accession No. NM_000201 (SEQ ID NO: 19)) and is produced after processing of the intercellular adhesion molecule 1 precursor polypeptide (Genbank Accession No.

NP_000192 (SEQ ID NO:20)).

2. Vascular Cell Adhesion Molecule-1 (VCAM-1)

[0144] VCAM-1 is a transmembrane cellular adhesion protein that mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium.

Upregulation of VCAM-1 in endothelial cells by cytokines occurs as a result of increased gene transcription {e.g., in response to Tumor necrosis factor-alpha (TNFa) and Interleukin- 1 (IL-1 )). VCAM-1 is encoded by the vascular cell adhesion molecule 1 gene (VCAM1 ;

Entrez GeneID:7412) and is produced after differential splicing of the transcript (Genbank Accession No. NM_001078 (variant 1 ; SEQ ID NO:21 ) or NM_080682 (variant 2)), and processing of the precursor polypeptide splice isoform (Genbank Accession No. NP_001069 (isoform a; SEQ ID NO:22) or NP_542413 (isoform b)).

[0145] In certain instances, the presence or level of an IgSF CAM is detected at the level of mRNA expression with an assay such as, for example, a hybridization assay or an amplification-based assay. In certain other instances, the presence or level of an IgSF CAM is detected at the level of protein expression using, for example, an immunoassay {e.g., ELISA) or an immunohistochemical assay. Suitable antibodies and/or ELISA kits for determining the presence or level of ICAM-1 and/or VCAM-1 in a sample such as a tissue sample, biopsy, serum, plasma, saliva, urine, or stool are available from, e.g., Invitrogen (Camarillo, CA), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and/or Abeam Inc. (Cambridge, MA).

K. Genetic Markers

[0146] The determination of the presence or absence of allelic variants in one or more genetic markers in a sample is also useful in the present invention. Non-limiting examples of genetic markers include, but are not limited to, any of the genes set forth in Tables 1 A- I E (e.g., a NOD2/CARD 15 gene, an IL12/IL23 pathway gene, etc.). Preferably, the presence or absence of at least one single nucleotide polymorphism (SNP) in the NOD2/CARD 15 gene and/or one or more genes in the IL12/IL23 pathway is determined. See, e.g., Barrett et al , Nat. Genet. , 40:955-62 (2008) and Wang et al. , Amer. J. Hum. Genet. , 84:399-405 (2009).

[0147] Table 1 A provides an exemplary list of IBD, UC, and CD genes wherein genotyping for the presence or absence of one or more allelic variants {e.g., SNPs) therein is useful in the prognostic methods of the present invention. Table IB provides additional exemplary genetic markers and corresponding SNPs that can be genotyped in accordance with the prognostic methods of the present invention. Tables 1 C- 1 E provide additional exemplary IBD, UC, and CD genetic markers and corresponding SNPs that can be genotyped in accordance with the prognostic methods described herein.

Table 1 A. IBD. CD & UC Genes

Table IB. IBP, CD & UC Genes & SNPs

IL18RI rs 1035127

CCR5

MAPK14 rs2237093

IL18 rsl 1214108

IFNG rs 10878698

^' MAP2 6 rs2905443

STAT4 rsl 584945

IL12A rs6800657

TYK2 rsl 2720356

ETV5 rs9867846

MAPK8 rsl7697885

Table 1C. CD Genes & SNPs

Gene SNP

NOD2 (R702W) rs2066844

NOD2 (G908R) rs2066845

NOD2 (3020insC) rs5743293

ATG16L1 (T300A) rs2241880

ATG16L1 rs3828309

IRGM rsl3361189

IRGM rs4958847

IRGM rslOOOl 13

IRGM rsl 1747270

TL1A/TNFSF15 rs6478109

TL1 A/TNFSF15 rs6478108

TL1 A/TNFSF15 rs4263839

PTN22 rs2476601

CCR6 rsl 456893

CCR6 rs2301436

5pl3/PTGER4 rsl 373692

5pl3/PTGER4 rs4495224

5pI3/PTGER4 rs7720838

5pl3PTGER4 rs4613763

ITLN1 rs2274910

ITLNI rs9286879

ITLN1 rsl 1584383

IBD5/5q31 rs2188962 IBD5/5q31 rs252057

IBD5/5q31 rs 10067603

GCKR rs780094

TNFRSF6B rs 1736135

ZNF365 rs224136

ZNF365 rs 10995271

C l l orf30 rs7927894

LRRK2;MUC19 rs l 175593

DLG5 rs2165047

IL-27 rs8049439

TLR2 rs4696480

TLR2 rs3804099

TLR2 rs3804100

TLR2 rs5743704

TLR2 rs2405432

TLR4 (D299G) rs4986790

TLR4 (T399I) rs4986791

TLR4 (S360N) rs4987233

TLR9 rs l 87084

TLR9 rs352140

NFC4 rs4821544

KIF21B rs l 1584383

IKZF1 rsl456893

C l l orOO rs7927894

CCL2,CCL7 rs991804

ICOSLG rs762421

TNFAIP3 rs7753394

FLJ45139 rs2836754

PTGER4 rs4613763

Table ID. UC Genes & SNPs

Table IE. IBD Genes & SNPs

[0148] Additional SNPs useful in the present invention include, e.g., rs2188962, rs9286879, rs 1 1584383, rs7746082, rs l 456893, rs l 551398, rsl 7582416, rs3764147, rs l 736135, rs4807569, rs7758080, and rs8098673. See, e.g., Barrett et al, Nat. Genet., 40:955-62 (2008).

1. NOD2/CARD15

[0149] The determination of the presence or absence of allelic variants such as SNPs in the NOD2/CARD 1 gene is particularly useful in the present invention. As used herein, the term "NOD2/CARD 15 variant" or "NOD2 variant" includes a nucleotide sequence of a NOD2 gene containing one or more changes as compared to the wild-type NOD2 gene or an amino acid sequence of a NOD2 polypeptide containing one or more changes as compared to the wild-type NOD2 polypeptide sequence. NOD2, also known as CARD15, has been localized to the IBDl locus on chromosome 16 and identified by positional-cloning (Hugot et al. , Nature, 41 1 :599-603 (2001 )) as well as a positional candidate gene strategy (Ogura et al , Nature, 41 1 :603-606 (2001 ); Hampe et al, Lancet, 357: 1925-1928 (2001)). The IBDl locus has a high multipoint linkage score (MLS) for inflammatory bowel disease (MLS=5.7 at marker D 16S41 1 in 16ql 2). See, e.g., Cho et al, Inflamm. Bowel Dis. , 3: 1 86-190 ( 1997); Akolkar et al , Am. J. Gastroenterol , 96: 1 127-1 132 (2001 ); Ohmen et al, Hum. Mol. Genet. , 5: 1679-1683 ( 1996); Parkes et al, Lancet, 348: 1588 ( 1996); Cavanaugh et αΙ., Αηη. Hum. Genet , 62:291 -8 ( 1998); Brant et al , Gastroenterology, 1 15: 1056-1061 (1998); Curran et al, Gastroenterology, 1 15: 1066- 1071 (1998); Hampe et al , Am. J. Hum. Genet. , 64:808-816 (1999); and Annese et al , Eur. J. Hum. Genet. , 7:567-573 (1999).

[0150] The mRNA (coding) and polypeptide sequences of human NOD2 are set forth in, e.g., Genbank Accession Nos. NM_022162 (SEQ ID NO:23) and NP_071445 (SEQ ID NO:24), respectively. In addition, the complete sequence of human chromosome 16 clone RP1 1 -327F22, which includes NOD2, is set forth in, e.g. , Genbank Accession No.

AC007728. Furthermore, the sequence of NOD2 from other species can be found in the GenBank database.

[0151] The NOD2 protein contains amino-terminal caspase recruitment domains (CARDs), which can activate NF-kappa B (NF-kB), and several carboxy-terminal leucine-rich repeat domains (Ogura et al, J. Biol Chem. , 276:4812-4818 (2001 )). NOD2 has structural homology with the apoptosis regulator Apaf-l/CED-4 and a class of plant disease resistant gene products (Ogura et al , supra). Similar to plant disease resistant gene products, NOD2 has an amino-terminal effector domain, a nucleotide-binding domain and leucine rich repeats (LRRs). Wild-type NOD2 activates nuclear factor NF-kappa B, making it responsive to bacterial lipopolysaccharides (LPS; Ogura et al , supra; Inohara et al. , 3. Biol. Chem., 276:2551 -2554 (2001 ). NOD2 can function as an intercellular receptor for LPS, with the leucine rich repeats required for responsiveness.

[0152] Variations at three single nucleotide polymorphisms in the coding region of NOD2 have been previously described. These three SNPs, designated R702W ("SNP 8"), G908R ("SNP 12"), and 1007fs ("SNP 13"), are located in the carboxy-terminal region of the NOD2 gene (Hugot et al. , supra). A further description of SNP 8, SNP 12, and SNP 13, as well as additional SNPs in the NOD2 gene suitable for use in the invention, can be found in, e.g., U.S. Patent Nos. 6,835,815; 6,858,391 ; and 7,592,437; and U.S. Patent Publication Nos. 20030190639, 20050054021 , and 20070072180.

[0153] In some embodiments, a NOD2 variant is located in a coding region of the NOD2 locus, for example, within a region encoding several leucine-rich repeats in the carboxy- terminal portion of the NOD2 polypeptide. Such NOD2 variants located in the leucine-rich repeat region of NOD2 include, without limitation, R702W ("SNP 8") and G908R ("SNP 12"). A NOD2 variant useful in the invention can also encode a NOD2 polypeptide with reduced ability to activate NF-kappa B as compared to NF-kappa B activation by a wild-type NOD2 polypeptide. As a non-limiting example, the NOD2 variant 1007fs ("SNP 13") results in a truncated NOD2 polypeptide which has reduced ability to induce NF-kappa B in response to LPS stimulation (Ogura et al. , Nature, 41 1 :603-606 (2001)).

[0154] A NOD2 variant useful in the invention can be, for example, R702W, G908R, or 1007fs. R702W, G908R, and 1007fs are located within the coding region of NOD2. In one embodiment, a method of the invention is practiced with the R702W NOD2 variant. As used herein, the term "R702W" includes a single nucleotide polymorphism within exon 4 of the NOD2 gene, which occurs within a triplet encoding amino acid 702 of the NOD2 protein. The wild-type NOD2 allele contains a cytosine (c) residue at position 138,991 of the AC007728 sequence, which occurs within a triplet encoding an arginine at amino acid702. The R702W NOD2 variant contains a thymine (t) residue at position 138,991 of the

AC007728 sequence, resulting in an arginine (R) to tryptophan (W) substitution at amino acid 702 of the NOD2 protein. Accordingly, this NOD2 variant is denoted "R702W" or "702W" and can also be denoted "R675W" based on the earlier numbering system of Hugot et al , supra. In addition, the R702W variant is also known as the "SNP 8" allele or a "2" allele at SNP 8. The NCBI SNP ID number for R702W or SNP 8 is rs2066844. As disclosed herein and described further below, the presence of the R702W NOD2 variant and other NOD2 variants can be conveniently detected, for example, by allelic discrimination assays or sequence analysis. Primers and probes specific for the R702W NOD2 variant can be found in Tables 3 and 4 in Example 6.

[0155] A method of the invention can also be practiced with the G908R NOD2 variant. As used herein, the term "G908R" includes a single nucleotide polymorphism within exon 8 of the NOD2 gene, which occurs within a triplet encoding amino acid 908 of the NOD2 protein. Amino acid 908 is located within the leucine rich repeat region of the NOD2 gene. The wild- type NOD2 allele contains a guanine (g) residue at position 128,377 of the AC007728 sequence, which occurs within a triplet encoding glycine at amino acid 908. The G908R NOD2 variant contains a cytosine (c) residue at position 128,377 of the AC007728 sequence, resulting in a glycine (G) to arginine (R) substitution at amino acid 908 of the NOD2 protein. Accordingly, this NOD2 variant is denoted "G908R" or "908R" and can also be denoted "G881R" based on the earlier numbering system of Hugot et al , supra. In addition, the G908R variant is also known as the "SNP 12" allele or a "2" allele at SNP 12. The NCBI SNP ID number for G908R SNP 12 is rs2066845. Primers and probes specific for the G908R NOD2 variant can be found in Tables 3 and 4 in Example 6.

[0156] A method of the invention can also be practiced with the 1007fs NOD2 variant. This variant is an insertion of a single nucleotide that results in a frame shift in the tenth leucine-rich repeat of the NOD2 protein and is followed by a premature stop codon. The resulting truncation of the NOD2 protein appears to prevent activation of NF-kappaB in response to bacterial lipopolysaccharides (Ogura et al, supra). As used herein, the term "1007fs" includes a single nucleotide polymorphism within exon 1 1 of the NOD2 gene, which occurs in a triplet encoding amino acid 1007 of the NOD2 protein. The 1007fs variant contains a cytosine which has been added at position 121 , 139 of the AC007728 sequence, resulting in a frame shift mutation at amino acid 1007. Accordingly, this NOD2 variant is denoted "1007fs" and can also be denoted "3020insC" or "980fs" based on the earlier numbering system of Hugot et al. , supra. In addition, the 1007fs NOD2 variant is also known as the "SNP 13" allele or a "2" allele at SNP 13. The NCBI SNP ID number for 1007fs or SNP 13 is rs2066847. Primers and probes specific for the 1007fs NOD2 variant can be found in Tables 3 and 4 in Example 6. [0157] One skilled in the art recognizes that a particular NOD2 variant allele or other polymorphic allele can be conveniently defined, for example, in comparison to a Centre d'Etude du Polymorphisme Humain (CEPH) reference individual such as the individual designated 1347-02 (Dib et al , Nature, 380: 152- 154 ( 1996)), using commercially available reference DNA obtained, for example, from PE Biosystems (Foster City, CA). In addition, specific information on SNPs can be obtained from the dbSNP of the National Center for Biotechnology Information (NCBI).

[0158] A NOD2 variant can also be located in a non-coding region of the NOD2 locus. Non-coding regions include, for example, intron sequences as well as 5' and 3' untranslated sequences. A non-limiting example of a NOD2 variant allele located in a non-coding region of the NOD2 gene is the JWl variant, which is described in Sugimura et al. , Am. J. Hum. Genet., 72:509-518 (2003) and U.S. Patent Publication No. 20070072180. Examples of NOD2 variant alleles located in the 3' untranslated region of the NOD2 gene include, without limitation, the JW15 and JWl 6 variant alleles, which are described in U.S. Patent Publication No. 20070072180. Examples of NOD2 variant alleles located in the 5' untranslated region (e.g., promoter region) of the NOD2 gene include, without limitation, the JW17 and JWl 8 variant alleles, which are described in U.S. Patent Publication No. 20070072180.

[0159] As used herein, the term "JW l variant allele" includes a genetic variation at nucleotide 158 of intervening sequence 8 (intron 8) of the NOD2 gene. In relation to the AC007728 sequence, the JWl variant allele is located at position 128, 143. The genetic variation at nucleotide 158 of intron 8 can be, but is not limited to, a single nucleotide substitution, multiple nucleotide substitutions, or a deletion or insertion of one or more nucleotides. The wild-type sequence of intron 8 has a cytosine at position 158. As non- limiting examples, a JWl variant allele can have a cytosine (c) to adenine (a), cytosine (c) to guanine (g), or cytosine (c) to thymine (t) substitution at nucleotide 158 of intron 8. In one embodiment, the JWl variant allele is a change from a cytosine (c) to a thymine (t) at nucleotide 158 of NOD2 intron 8.

[0160] The term "JW15 variant allele" includes a genetic variation in the 3' untranslated region of NOD2 at nucleotide position 1 18,790 of the AC007728 sequence. The genetic variation at nucleotide 1 18,790 can be, but is not limited to, a single nucleotide substitution, multiple nucleotide substitutions, or a deletion or insertion of one or more nucleotides. The wild-type sequence has an adenine (a) at position 1 18,790. As non-limiting examples, a JW l 5 variant allele can have an adenine (a) to cytosine (c), adenine (a) to guanine (g), or adenine (a) to thymine (t) substitution at nucleotide 1 1 8,790. In one embodiment, the JW l 5 variant allele is a change from an adenine (a) to a cytosine (c) at nucleotide 1 18,790.

[0161] As used herein, the term "JW 16 variant allele" includes a genetic variation in the 3 ' untranslated region of NOD2 at nucleotide position 1 18,03 1 of the AC007728 sequence. The genetic variation at nucleotide 1 1 8,031 can be, but is not limited to, a single nucleotide substitution, multiple nucleotide substitutions, or a deletion or insertion of one or more nucleotides. The wild-type sequence has a guanine (g) at position 1 18,031 . As non-limiting examples, a JW l 6 variant allele can have a guanine (g) to cytosine (c), guanine (g) to adenine (a), or guanine (g) to thymine (t) substitution at nucleotide 1 18,031 . In one embodiment, the JW l 6 variant allele is a change from a guanine (g) to an adenine (a) at nucleotide 1 18,031 .

[0162] The term "JW 17 variant allele" includes a genetic variation in the 5' untranslated region of NOD2 at nucleotide position 154,688 of the AC007728 sequence. The genetic variation at nucleotide 154,688 can be, but is not limited to, a single nucleotide substitution, multiple nucleotide substitutions, or a deletion or insertion of one or more nucleotides. The wild-type sequence has a cytosine (c) at position 154,688. As non-limiting examples, a JW l 7 variant allele can have a cytosine (c) to guanine (g), cytosine (c) to adenine (a), or cytosine (c) to thymine (t) substitution at nucleotide 154,688. In one embodiment, the JW l 7 variant allele is a change from a cytosine (c) to a thymine (t) at nucleotide 154,688.

[0163] As used herein, the term "JW l 8 variant allele" includes a genetic variation in the 5' untranslated region of NOD2 at nucleotide position 154,471 of the AC007728 sequence. The genetic variation at nucleotide 154,471 can be, but is not limited to, a single nucleotide substitution, multiple nucleotide substitutions, or a deletion or insertion of one or more nucleotides. The wild-type sequence has a cytosine (c) at position 154,471 . As non-limiting examples, a JWl 8 variant allele can have a cytosine (c) to guanine (g), cytosine (c) to adenine (a), or cytosine (c) to thymine (t) substitution at nucleotide 154,471 . In one embodiment, the JW l 8 variant allele is a change from a cytosine (c) to a thymine (t) at nucleotide 154,471 .

[0164] It is understood that the methods of the invention can be practiced with these or other NOD2 variant alleles located in a coding region or non-coding region (e.g., intron or promoter region) of the NOD2 locus. It is further understood that the methods of the invention can involve determining the presence of one, two, three, four, or more NOD2 variants, including, but not limited to, the SNP 8, SNP 12, and SNP 13 alleles, and other coding as well as non-coding region variants.

2. miRNAs

[0165] Generally, microRNAs (miRNA) are single-stranded RNA molecules of about 21 - 23 nucleotides in length which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed, but miRNAs are not translated into protein (non- coding RNA). Instead, each primary transcript (a pri-miRNA) is processed into a short stem- loop structure called a pre-miRNA and finally into a functional mature miRNA. Mature miRNA molecules are either partially or completely complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression. The identification of miRNAs is described, e.g., in Lagos-Quintana et al. , Science, 294:853-858; Lau et al , Science, 294:858-862; and Lee et al , Science, 294:862-864.

[0166] Mammalian miRs are usually complementary to a site in the 3' UTR of the target mRNA sequence. The annealing of the miRNA to the target mRNA inhibits protein translation by blocking the protein translation machinery or facilitates the cleavage and degradation of the target mRNA through a process similar to RNA interference (RNAi). miRNAs may also target methylation of genomic sites which correspond to targeted mRNAs.

[0167] In some embodiments, the prognostic marker of the invention comprises at least one miRNA sequence {e.g., pre-miRNA or mature miRNA). In preferred embodiments, the miRNA sequence targets the expression of any of the biochemical, serological, or genetic markers described herein, e.g., cytokines, growth factors, acute phase proteins,

apolipoproteins, defensins, cadherins; or any of the genes set forth in Tables 1 A-1E (e.g., NOD2). Generally, the presence or level of the miRNA sequence of interest is detected in an individual's sample and included in the prognostic marker profile to aid in the prediction of whether a patient will have a particular risk of developing postoperative complications after pouch surgery. Exemplary miRNA sequences suitable for detection as prognostic markers in accordance with the invention are listed in Table 2. Table 2

hsa-miR-153 (ΜΪΜΑΤ0000439): hsa-miR-760 (MIMAT0004957): hsa-miR-106a* ( IMAT0004517); hsa- miR-371 -5p (MI AT0004687): hsa-miR-376a (MI AT0000729): hsa-miR-144 ( I AT0000436); hsa- miR-5 l 8c* ( I AT0002847): hsa-miR-548d-5p (MI AT00048 I 2): hsa-miR-365 (MIMAT00007 I0); hsa- miR-548c-5p (MIMAT0004806); hsa-miR-587 ( I AT0003253); hsa-miR-33a* (MIMAT0004506); hsa- miR-574-3p (MIMAT0003239); hsa-miR-568 (MI AT0003232); hsa-let-7i ( IMAT0000415); hsa-miR- 148b* (MI AT0004699); hsa-miR-655 (M1MAT00033 1 ): hsa-miR-548a-5p (MIMAT0004803); hsa-miR- 148a* (MIMAT0004549): hsa-miR-61 (MIMAT0003281 ): hsa-miR-146b-3p (MI AT0004766); hsa-miR- 149 (MIMAT0000450); hsa-miR-217 ( 1 AT0000274); hsa-miR- 196b (MIMAT0001080); hsa-miR-22* (M1MAT0004495); hsa-miR-1 7 ( 1 AT0000429): hsa-miR-498 (MIMAT0002824); hsa-let-7g (MIMAT0000414); hsa-miR-155 (MIMAT0000646): hsa-miR-383 ( IMAT0000738); hsa-miR-576-3p ( I AT0004796); hsa-miR-183* (MlMAT0004i60): hsa-miR-555 (MIMAT0003219); hsa-miR-589 ( IMAT0004799); hsa-miR-338-5p (MIMAT0004701 ); hsa-miR-522 (MIMAT0002868); hsa-miR-643 (MIMAT000331 ); hsa-miR-369-3p (MIMAT0000721 ): hsa-miR-552 (ΜΪΜΑΤ0003215); hsa-miR-499-5p (MI AT0002870); hsa-miR-137 (MI AT0000429); hsa-miR-338-5p (M1 AT0004701 ); hsa-miR-374b (MIMAT0004955); hsa-miR-376c (M1 AT0000720): hsa-miR-588 (MIMAT0003255); hsa-miR-212 (MI AT0000269); hsa-miR-1 2 (MIMAT0000426)

E-cadherin hsa-miR-143* (MIMAT0004599); hsa-miR-544 (M1 AT0003164); hsa-miR-920 (MIMAT0004970); hsa- miR-635 (MIMAT0003305); hsa-miR-340* (MI AT0000750); hsa-miR-665 (MI AT0004952); hsa-miR- 217 (MIMAT0000274); hsa-miR-9* (MIMAT0000442); hsa-miR-612 (MI AT0003280); hsa-miR-920 (MIMAT0004970); hsa-miR-382 (MIMAT0000737); hsa-miR-340 (MIMAT0004692); hsa-miR-34c-3p (MIMAT0004677); hsa-miR-1 (MIMAT0000416); hsa-miR-571 ( IMAT0003236); hsa-miR-499-3p (MIMAT0004772); hsa-miR-708* (MI AT0004927); hsa-miR-220b ( I AT0004908); hsa-miR-206 (MIMAT0000462); hsa-miR-92a (M1MAT0000092); hsa-miR-92b (MIMAT0003218); hsa-miR-217 (M1MAT0000274)

NOD2 hsa-miR-671-5p (MIMAT0003880); hsa-miR-20a* (MIMAT000449 ); hsa-miR-124 (MI AT0000422);

hsa-miR-122 (MIMAT0000421); hsa-miR-192 (MI AT0000222); hsa-miR-215 (MIMAT0000272); hsa- miR-495 (MIMAT0002817); hsa-miR-342-5p (M1 AT0004694); hsa-miR-512-5p (MIMAT0002822); hsa- miR-453 (MIMAT0001630); hsa-miR-215 (MIMAT0000272); hsa-miR-192 (MIMAT0000222)

The Accession Nos. for the mature miRNA sequences correspond to entries that can be found in the miRBase Sequence Database from the Sanger Institute. The miRBase Sequence Database is a searchable database of published miRNA sequences and annotation. The miRBase Sequence Database Accession Nos. are herein incorporated by reference in their entirety for all purposes.

[0168] In certain embodiments, the miR set forth in Table 2 is between about 17 to 25 nucleotides in length and comprises a sequence that is at least 90% identical to a miRNA set forth in the listed Accession No. for the mature miRNA sequence. In certain embodiments, a miRNA is 17, 18, 19, 20, 21 , 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. Moreover, in certain embodiments, a miR has a sequence that is or is at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.1 , 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% identical to the miRNA in Table 2.

[0169] In some therapeutic embodiments, the complement of the miR set forth in Table 2 is useful. This is known as a miRNA inhibitor. A miRNA inhibitor is between about 17 to 25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of a mature miRNA. In certain embodiments, a miRNA inhibitor molecule is 17, 18, 19, 20, 21 , 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. Moreover, a miR inhibitor has a sequence (from 5' to 3') that is or is at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.1 , 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any range derivable therein, to the 5' to 3' sequence of a mature miRNA. L. Other Prognostic Markers

[0170] Additional prognostic markers suitable for use in the present invention include, but are not limited to, lactoferrin, anti-lactoferrin antibodies, elastase, calprotectin, hemoglobin, and combinations thereof.

[0171] The determination of the presence or level of lactoferrin in a sample is also useful in the present invention. In certain instances, the presence or level of lactoferrin is detected at the level of mRNA expression with an assay such as, for example, a hybridization assay or an amplification-based assay. In certain other instances, the presence or level of lactoferrin is detected at the level of protein expression using, for example, an immunoassay (e.g., ELISA) or an immunohistochemical assay. An ELISA kit available from Calbiochem (San Diego, CA) can be used to detect human lactoferrin in a plasma, urine, bronchoalveolar lavage, or cerebrospinal fluid sample. Similarly, an ELISA kit available from U.S. Biological

(Swampscott, MA) can be used to determine the level of lactoferrin in a plasma sample. Likewise, ELISA kits available from TECHLAB, Inc. (Blacksburg, VA) can be used to determine the level of lactoferrin in a stool sample. Additionally, U.S. Patent Publication No. 20040137536 describes an ELISA assay for determining the presence of elevated lactoferrin levels in a stool sample, and U.S. Patent Publication No. 20040033537 describes an ELISA assay for determining the concentration of endogenous lactoferrin in a stool, mucus, or bile sample. In some embodiments, then presence or level of anti-lactoferrin antibodies can be detected in a sample using, e.g., lactoferrin protein or a fragment thereof.

[0172] In addition, hemoccult, fecal occult blood, is often indicative of gastrointestinal illness and various kits have been developed to monitor gastrointestinal bleeding. For example, Hemoccult SENSA, a Beckman Coulter product, is a diagnostic aid for

gastrointestinal bleeding, iron deficiency, peptic ulcers, ulcerative colitis, and, in some instances, in screening for colorectal cancer. This particular assay is based on the oxidation of guaiac by hydrogen peroxide to produce a blue color. A similar colorimetric assay is commercially available from Helena Laboratories (Beaumont, TX) for the detection of blood in stool samples. Other methods for detecting occult blood in a stool sample by determining the presence or level of hemoglobin or heme activity are described in, e.g., U.S. Patent Nos. 4,277,250, 4,920,045, 5,081 ,040, and 5,310,684.

[0173] Calprotectin is a calcium and zinc-binding protein found in all cells, tissues, and fluids in the body. Calprotectin is a major protein in neutrophilic granulocytes and macrophages and accounts for as much as 60% of the total protein in the cytosolic fraction of these cells. It is therefore a surrogate marker of neutrophil turnover. Its concentration in stool correlates with the intensity of neutrophil infiltration of the intestinal mucosa and with the severity of inflammation. Calprotectin can be measured with an ELISA using small (50- 100 mg) fecal samples {see, e.g., Johne et al, Scand J Gastroenterol., 36:291 -296 (2001 )).

VI. Assays

[0174] Any of a variety of assays, techniques, and kits known in the art can be used to detect or determine the presence or level of one or more markers in a sample to predict whether a patient will have a particular risk or probability of developing postoperative complications following pouch surgery.

[0175] The present invention relies, in part, on determining the presence or level of at least one marker in a sample obtained from an individual. As used herein, the term "detecting the presence of at least one marker" includes determining the presence of each marker of interest by using any quantitative or qualitative assay known to one of skill in the art. In certain instances, qualitative assays that determine the presence or absence of a particular trait, variable, or biochemical or serological substance {e.g., protein or antibody) are suitable for detecting each marker of interest. In certain other instances, quantitative assays that determine the presence or absence of RNA, protein, antibody, or activity are suitable for detecting each marker of interest. As used herein, the term "detecting the level of at least one marker" includes determining the level of each marker of interest by using any direct or indirect quantitative assay known to one of skill in the art. In certain instances, quantitative assays that determine, for example, the relative or absolute amount of RNA, protein, antibody, or activity are suitable for detecting the level of each marker of interest. One skilled in the art will appreciate that any assay useful for detecting the level of a marker is also useful for detecting the presence or absence of the marker.

[0176] As used herein, the term "antibody" includes a population of immunoglobulin molecules, which can be polyclonal or monoclonal and of any isotype, or an immunologically active fragment of an immunoglobulin molecule. Such an immunologically active fragment contains the heavy and light chain variable regions, which make up the portion of the antibody molecule that specifically binds an antigen. For example, an immunologically active fragment of an immunoglobulin molecule known in the art as Fab, Fab' or F(ab')₂ is included within the meaning of the term antibody. [0177] Flow cytometry can be used to detect the presence or level of one or more markers in a sample. Such flow cytometric assays, including bead based immunoassays, can be used to determine, e.g., antibody marker levels in the same manner as described for detecting serum antibodies to Candida albicans and HIV proteins {see, e.g., Bishop and Davis, J. Immunol. Methods, 210:79-87 (1997); McHugh et al , J. Immunol. Methods, 1 16:213 ( 1989); Scillian et al , Blood, 73:2041 (1989)).

[0178] Phage display technology for expressing a recombinant antigen specific for a marker can also be used to detect the presence or level of one or more markers in a sample. Phage particles expressing an antigen specific for, e.g., an antibody marker can be anchored, if desired, to a multi-well plate using an antibody such as an anti-phage monoclonal antibody (Felici et al , "Phage-Displayed Peptides as Tools for Characterization of Human Sera" in Abelson (Ed.), Methods in Enzymol , 267, San Diego: Academic Press, Inc. (1996)).

[0179] A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used to detect the presence or level of one or more markers in a sample {see, e.g., Self and Cook, Curr. Opin. Biotechnol, 7:60-65 (1996)). The term immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), antigen capture ELISA, sandwich ELISA, IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction with laser induced fluorescence {see, e.g., Schmalzing and Nashabeh, Electrophoresis, 18:2184-2193 (1997); Bao, /. Chromatogr. B. Biomed. Sci., 699:463-480 (1997)). Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention {see, e.g., Rongen et al. , J. Immunol. Methods, 204: 105-133 (1997)). In addition, nephelometry assays, in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the present invention. Nephelometry assays are commercially available from Beckman Coulter (Brea, CA; Kit #449430) and can be performed using a Behring Nephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biol. Chem., 27:261 -276 (1989)). [0180] Antigen capture ELISA can be useful for detecting the presence or level of one or more markers in a sample. For example, in an antigen capture ELISA, an antibody directed to a marker of interest is bound to a solid phase and sample is added such that the marker is bound by the antibody. After unbound proteins are removed by washing, the amount of bound marker can be quantitated using, e.g., a radioimmunoassay (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988)). Sandwich ELISA can also be suitable for use in the present invention. For example, in a two-antibody sandwich assay, a first antibody is bound to a solid support, and the marker of interest is allowed to bind to the first antibody. The amount of the marker is quantitated by measuring the amount of a second antibody that binds the marker. The antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), pieces of a solid substrate material or membrane (e.g., plastic, nylon, paper), and the like. An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.

[0181] A radioimmunoassay using, for example, an iodine- 125 (^{l 2:}T) labeled secondary antibody (Harlow and Lane, supra) is also suitable for detecting the presence or level of one or more markers in a sample. A secondary antibody labeled with a chemiluminescent marker can also be suitable for use in the present invention. A chemiluminescence assay using a chemiluminescent secondary antibody is suitable for sensitive, non-radioactive detection of marker levels. Such secondary antibodies can be obtained commercially from various sources, e.g., Amersham Lifesciences, Inc. (Arlington Heights, IL).

[0182] The immunoassays described above are particularly useful for detecting the presence or level of one or more markers in a sample. As a non-limiting example, a fixed neutrophil ELISA is useful for determining whether a sample is positive for ANCA or for determining ANCA levels in a sample. Similarly, an ELISA using yeast cell wall phosphopeptidomannan is useful for determining whether a sample is positive for ASCA-IgA and/or ASCA-IgG, or for determining ASCA-IgA and/or ASCA-IgG levels in a sample. An ELISA using OmpC protein or a fragment thereof is useful for determining whether a sample is positive for anti-OmpC antibodies, or for determining anti-OmpC antibody levels in a sample. An ELISA using 12 protein or a fragment thereof is useful for determining whether a sample is positive for anti-I2 antibodies, or for determining anti-I2 antibody levels in a sample. An ELISA using flagellin protein (e.g., Cbir- 1 flagellin) or a fragment thereof is useful for determining whether a sample is positive for anti-flagellin antibodies, or for determining anti-flagellin antibody levels in a sample. In addition, the immunoassays described above are particularly useful for detecting the presence or level of other markers in a sample.

[0183] Specific immunological binding of the antibody to the marker of interest can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. An antibody labeled with iodine- 125 (^{l 25}I) can be used for determining the levels of one or more markers in a sample. A chemiluminescence assay using a chemiluminescent antibody specific for the marker is suitable for sensitive, non-radioactive detection of marker levels. An antibody labeled with fluorochrome is also suitable for determining the levels of one or more markers in a sample. Examples of fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and lissamine. Secondary antibodies linked to fluorochromes can be obtained commercially, e.g., goat F(ab')? anti-human IgG-FITC is available from Tago Immunologicals (Burlingame, CA).

[0184] Indirect labels include various enzymes well-known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, urease, and the like. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p-nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-P-D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm. An urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals; St. Louis, MO). A useful secondary antibody linked to an enzyme can be obtained from a number of commercial sources, e.g., goat F(ab')₂ anti-human IgG-alkaline phosphatase can be purchased from Jackson ImmunoResearch (West Grove, PA.).

[0185] A signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect

1 ^5

radiation such as a gamma counter for detection of ^" I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked antibodies, a quantitative analysis of the amount of marker levels can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, CA) in accordance with the manufacturer's instructions. If desired, the assays described herein can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.

[0186] Quantitative Western blotting can also be used to detect or determine the presence or level of one or more markers in a sample. Western blots can be quantitated by well-known methods such as scanning densitometry or phosphorimaging. As a non-limiting example, protein samples are electrophoresed on 10% SDS-PAGE Laemmli gels. Primary murine monoclonal antibodies are reacted with the blot, and antibody binding can be confirmed to be linear using a preliminary slot blot experiment. Goat anti-mouse horseradish peroxidase- coupled antibodies (BioRad) are used as the secondary antibody, and signal detection performed using chemiluminescence, for example, with the Renaissance chemiluminescence kit (New England Nuclear; Boston, MA) according to the manufacturer' s instructions.

Autoradiographs of the blots are analyzed using a scanning densitometer (Molecular Dynamics; Sunnyvale, CA) and normalized to a positive control. Values are reported, for example, as a ratio between the actual value to the positive control (densitometric index). Such methods are well known in the art as described, for example, in Parra et al, J. Vase. Surg. , 28:669-675 (1998).

[0187] Alternatively, a variety of immunohistochemical assay techniques can be used to detect or determine the presence or level of one or more markers in a sample. The term "immunohistochemical assay" encompasses techniques that utilize the visual detection of fluorescent dyes or enzymes coupled (i.e., conjugated) to antibodies that react with the marker of interest using fluorescent microscopy or light microscopy and includes, without limitation, direct fluorescent antibody assay, indirect fluorescent antibody (IFA) assay, anticomplement immunofluorescence, avidin-biotin immunofluorescence, and

immunoperoxidase assays. An IFA assay, for example, is useful for determining whether a sample is positive for ANCA, the level of ANCA in a sample, whether a sample is positive for pANCA, the level of pANCA in a sample, and/or an ANCA staining pattern (e.g., cANCA, pANCA, NSNA, and/or SAPPA staining pattern). The concentration of ANCA in a sample can be quantitated, e.g., through endpoint titration or through measuring the visual intensity of fluorescence compared to a known reference standard. [0188] Alternatively, the presence or level of a marker of interest can be determined by detecting or quantifying the amount of the purified marker. Purification of the marker can be achieved, for example, by high pressure liquid chromatography (HPLC), alone or in combination with mass spectrometry (e.g., MALDI/MS, MALDI-TOF/MS, SELDI-TOF/MS, tandem MS, etc.). Qualitative or quantitative detection of a marker of interest can also be determined by well-known methods including, without limitation, Bradford assays,

Coomassie blue staining, silver staining, assays for radiolabeled protein, and mass spectrometry.

[0189] The analysis of a plurality of markers may be carried out separately or

simultaneously with one test sample. For separate or sequential assay of markers, suitable apparatuses include clinical laboratory analyzers such as the ElecSys (Roche), the AxSym (Abbott), the Access (Beckman), the ADVIA^®, the CENTAUR^® (Bayer), and the NICHOLS ADVANTAGE^® (Nichols Institute) immunoassay systems. Preferred apparatuses or protein chips perform simultaneous assays of a plurality of markers on a single surface. Particularly useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different markers. Such formats include protein

microarrays, or "protein chips" (see, e.g., Ng et al. , J. Cell Mol. Med., 6:329-340 (2002)) and certain capillary devices (see, e.g., U.S. Pat. No. 6,019,944). In these embodiments, each discrete surface location may comprise antibodies to immobilize one or more markers for detection at each location. Surfaces may alternatively comprise one or more discrete particles (e.g., microparticles or nanoparticles) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one or more markers for detection.

[0190] In addition to the above-described assays for detecting the presence or level of various markers of interest, analysis of marker mRNA levels using routine techniques such as Northern analysis, reverse-transcriptase polymerase chain reaction (RT-PCR), or any other methods based on hybridization to a nucleic acid sequence that is complementary to a portion of the marker coding sequence (e.g., slot blot hybridization) are also within the scope of the present invention. Applicable PCR amplification techniques are described in, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. New York ( 1999), Chapter 7 and Supplement 47; Theophilus et al, "PCR Mutation Detection Protocols," Humana Press, (2002); and Innis et al., PCR Protocols, San Diego, Academic Press, Inc. (1990). General nucleic acid hybridization methods are described in Anderson, "Nucleic Acid Hybridization," BIOS Scientific Publishers, 1999. Amplification or hybridization of a plurality of transcribed nucleic acid sequences (e.g., mRNA or cDNA) can also be performed from mRNA or cDNA sequences arranged in a microarray. Microarray methods are generally described in Hardiman, "Microarrays Methods and Applications: Nuts & Bolts," DNA Press, 2003; and Baldi et al., "DNA Microarrays and Gene Expression: From

Experiments to Data Analysis and Modeling," Cambridge University Press, 2002.

[0191] Several markers of interest may be combined into one test for efficient processing of a multiple of samples. In addition, one skilled in the art would recognize the value of testing multiple samples (e.g., at successive time points, etc.) from the same subject. Such testing of serial samples can allow the identification of changes in marker levels over time. Increases or decreases in marker levels, as well as the absence of change in marker levels, can also provide useful prognostic and predictive information to facilitate treatment.

[0192] A panel for measuring one or more of the markers described above may be constructed to provide relevant information related to the approach of the invention for predicting or determining a risk of developing dysplasia/cancer and/or an inflammatory complication (e.g., pouchitis) in an individual following pouch surgery (e.g., IPAA). Such a panel may be constructed to detect or determine the presence or level of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 1 8, 19, 20, 25, 30, 35, 40, or more individual markers. The analysis of a single marker or subsets of markers can also be carried out by one skilled in the art in various clinical settings. These include, but are not limited to, ambulatory, urgent care, critical care, intensive care, monitoring unit, inpatient, outpatient, physician office, medical clinic, and health screening settings.

[0193] The analysis of markers could be carried out in a variety of physical formats as well. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate treatment, diagnosis, and prognosis in a timely fashion.

[0194] In view of the above, one skilled in the art realizes that the methods of the invention for providing prognostic and predictive information regarding post-surgery risk associated with pouch surgery (e.g., by determining the presence or concentration level of one or more markers described herein) can be practiced using one or any combination of the well-known assays described above or other assays known in the art. VII. Methods of Genotyping

[0195] A variety of means can be used to genotype an individual at a polymorphic site in the NOD2 gene or any other genetic marker described herein to determine whether a sample (e.g., a nucleic acid sample) contains a specific variant allele or haplotype. For example, enzymatic amplification of nucleic acid from an individual can be conveniently used to obtain nucleic acid for subsequent analysis. The presence or absence of a specific variant allele or haplotype in one or more genetic markers of interest can also be determined directly from the individual's nucleic acid without enzymatic amplification. In certain preferred embodiments, an individual is genotyped at the NOD2 locus.

[0196] Genotyping of nucleic acid from an individual, whether amplified or not, can be performed using any of various techniques. Useful techniques include, without limitation, polymerase chain reaction (PCR) based analysis, sequence analysis, and electrophoretic analysis, which can be used alone or in combination. As used herein, the term "nucleic acid" means a polynucleotide such as a single- or double-stranded DNA or RNA molecule including, for example, genomic DNA, cDNA and mRNA. This term encompasses nucleic acid molecules of both natural and synthetic origin as well as molecules of linear, circular, or branched configuration representing either the sense or antisense strand, or both, of a native nucleic acid molecule. It is understood that such nucleic acids can be unpurified, purified, or attached, for example, to a synthetic material such as a bead or column matrix.

[0197] Material containing nucleic acid is routinely obtained from individuals. Such material is any biological matter from which nucleic acid can be prepared. As non-limiting examples, material can be whole blood, serum, plasma, saliva, cheek swab, sputum, or other bodily fluid or tissue that contains nucleic acid. In one embodiment, a method of the present invention is practiced with whole blood, which can be obtained readily by non-invasive means and used to prepare genomic DNA. In another embodiment, genotyping involves amplification of an individual's nucleic acid using the polymerase chain reaction (PCR). Use of PCR for the amplification of nucleic acids is well known in the art (see, e.g., Mullis et al. (Eds.), The Polymerase Chain Reaction, Birkhauser, Boston, (1994)). In yet another embodiment, PCR amplification is performed using one or more fluorescently labeled primers. In a further embodiment, PCR amplification is performed using one or more labeled or unlabeled primers that contain a DNA minor groove binder. [0198] Any of a variety of different primers can be used to amplify an individual's nucleic acid by PCR in order to determine the presence or absence of a variant allele in the NOD2 gene or other genetic marker in a method of the invention. For example, the PCR primers listed in Table 3 (SEQ ID NOS:25-32) can be used to amplify specific regions of the NOD2 locus. As non-limiting examples, the region surrounding R702W ("SNP 8") can be amplified using SEQ ID NOS: 27 and 28, G908R ("SNP 12") can be amplified using SEQ ID NOS: 29 and 30, and the region surrounding 1007fs ("SNP 13") can be amplified using SEQ ID NOS: 31 and 32. As understood by one skilled in the art, additional primers for PCR analysis can be designed based on the sequence flanking the polymorphic site(s) of interest in the NOD2 gene or other genetic marker. As a non-limiting example, a sequence primer can contain from about 15 to about 30 nucleotides of a sequence upstream or downstream of the polymorphic site of interest in the NOD2 gene or other genetic marker. Such primers generally are designed to have sufficient guanine and cytosine content to attain a high melting temperature which allows for a stable annealing step in the amplification reaction. Several computer programs, such as Primer Select, are available to aid in the design of PCR primers.

[0199] A Taqman^® allelic discrimination assay available from Applied Biosystems can be useful for genotyping an individual at a polymorphic site and thereby determining the presence or absence of a particular variant allele or haplotype in the NOD2 gene or other genetic marker described herein. In a Taqman^® allelic discrimination assay, a specific fluorescent dye-labeled probe for each allele is constructed. The probes contain different fluorescent reporter dyes such as FAM and VIC to differentiate amplification of each allele. In addition, each probe has a quencher dye at one end which quenches fluorescence by fluorescence resonance energy transfer. During PCR, each probe anneals specifically to complementary sequences in the nucleic acid from the individual. The 5' nuclease activity of Taq polymerase is used to cleave only probe that hybridizes to the allele. Cleavage separates the reporter dye from the quencher dye, resulting in increased fluorescence by the reporter dye. Thus, the fluorescence signal generated by PCR amplification indicates which alleles are present in the sample. Mismatches between a probe and allele reduce the efficiency of both probe hybridization and cleavage by Taq polymerase, resulting in little to no fluorescent signal. Those skilled in the art understand that improved specificity in allelic discrimination assays can be achieved by conjugating a DNA minor groove binder (MGB) group to a DNA probe as described, e.g., in Kutyavin et at, Nuc. Acids Research 28:655-661 (2000). Minor groove binders include, but are not limited to, compounds such as dihydrocyclopyrroloindole tripeptide (DPI3). Exemplary Taqman " probes suitable for detecting the SNP 8, SNP 12, and SNP 13 allelic variants in the NOD2 gene are set forth in Table 4 (SEQ ID NOS:33-42).

[0200] Sequence analysis can also be useful for genotyping an individual according to the methods described herein to determine the presence or absence of a particular variant allele or haplotype in the NOD2 gene or other genetic marker. As is known by those skilled in the art, a variant allele of interest can be detected by sequence analysis using the appropriate primers, which are designed based on the sequence flanking the polymorphic site of interest in the NOD2 gene or other genetic marker. For example, a NOD2 variant allele can be detected by sequence analysis using primers disclosed herein, e.g., the PCR primers set forth in Table 3 (SEQ ID NOS:25-32). Additional or alternative sequence primers can contain from about 15 to about 30 nucleotides of a sequence that corresponds to a sequence about 40 to about 400 base pairs upstream or downstream of the polymorphic site of interest in the NOD2 gene or other genetic marker. Such primers are generally designed to have sufficient guanine and cytosine content to attain a high melting temperature which allows for a stable annealing step in the sequencing reaction.

[0201] The term "sequence analysis" includes any manual or automated process by which the order of nucleotides in a nucleic acid is determined. As an example, sequence analysis can be used to determine the nucleotide sequence of a sample of DNA. The term sequence analysis encompasses, without limitation, chemical and enzymatic methods such as dideoxy enzymatic methods including, for example, Maxam-Gilbert and Sanger sequencing as well as variations thereof. The term sequence analysis further encompasses, but is not limited to, capillary array DNA sequencing, which relies on capillary electrophoresis and laser-induced fluorescence detection and can be performed using instruments such as the MegaB ACE 1000 or ABI 3700. As additional non-limiting examples, the term sequence analysis encompasses thermal cycle sequencing (see, Sears et al., Biotechniques 13:626-633 ( 1992)); solid-phase sequencing (see, Zimmerman et al, Methods Mol. Cell Biol. 3:39-42 ( 1992); and sequencing with mass spectrometry, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (see, MALDI-TOF MS; Fu et al, Nature Biotech. 16:381 -384 ( 1998)). The term sequence analysis further includes, but is not limited to, sequencing by

hybridization (SBH), which relies on an array of all possible short oligonucleotides to identify a segment of sequence (see, Chee et al, Science 274:610-614 ( 1996); Drmanac et al, Science 260: 1649- 1652 (1993); and Drmanac et al, Nature Biotech. 16:54-58 (1998)). One skilled in the art understands that these and additional variations are encompassed by the term sequence analysis as defined herein.

[0202] Electrophoretic analysis also can be useful in genotyping an individual according to the methods of the present invention to determine the presence or absence of a particular variant allele or haplotype in the NOD2 gene or other genetic marker. "Electrophoretic analysis" as used herein in reference to one or more nucleic acids such as amplified fragments includes a process whereby charged molecules are moved through a stationary medium under the influence of an electric field. Electrophoretic migration separates nucleic acids primarily on the basis of their charge, which is in proportion to their size, with smaller molecules migrating more quickly. The term electrophoretic analysis includes, without limitation, analysis using slab gel electrophoresis, such as agarose or polyacrylamide gel electrophoresis, or capillary electrophoresis. Capillary electrophoretic analysis generally occurs inside a small-diameter (50-100 m) quartz capillary in the presence of high (kilovolt- level) separating voltages with separation times of a few minutes. Using capillary electrophoretic analysis, nucleic acids are conveniently detected by UV absorption or fluorescent labeling, and single-base resolution can be obtained on fragments up to several hundred base pairs. Such methods of electrophoretic analysis, and variations thereof, are well known in the art, as described, for example, in Ausubel et al., Current Protocols in Molecular Biology Chapter 2 (Supplement 45) John Wiley & Sons, Inc. New York (1999).

[0203] Restriction fragment length polymorphism (RFLP) analysis can also be useful for genotyping an individual according to the methods of the present invention to determine the presence or absence of a particular variant allele or haplotype in the NOD2 gene or other genetic marker {see, Jarcho et al. in Dracopoli et al., Current Protocols in Human Genetics pages 2.7.1 -2.7.5, John Wiley & Sons, New York; Innis et al , (Ed.), PCR Protocols, San Diego: Academic Press, Inc. (1990)). As used herein, "restriction fragment length polymorphism analysis" includes any method for distinguishing polymorphic alleles using a restriction enzyme, which is an endonuclease that catalyzes degradation of nucleic acid following recognition of a specific base sequence, generally a palindrome or inverted repeat. One skilled in the art understands that the use of RFLP analysis depends upon an enzyme that can differentiate a variant allele from a wild-type or other allele at a polymorphic site.

[0204] In addition, allele-specific oligonucleotide hybridization can be useful for genotyping an individual in the methods described herein to determine the presence or absence of a particular variant allele or haplotype in the NOD2 gene or other genetic marker. Allele-specific oligonucleotide hybridization is based on the use of a labeled oligonucleotide probe having a sequence perfectly complementary, for example, to the sequence

encompassing the variant allele. Under appropriate conditions, the variant allele-specific probe hybridizes to a nucleic acid containing the variant allele but does not hybridize to the one or more other alleles, which have one or more nucleotide mismatches as compared to the probe. If desired, a second allele-specific oligonucleotide probe that matches an alternate (e.g., wild-type) allele can also be used. Similarly, the technique of allele-specific oligonucleotide amplification can be used to selectively amplify, for example, a variant allele by using an allele-specific oligonucleotide primer that is perfectly complementary to the nucleotide sequence of the variant allele but which has one or more mismatches as compared to other alleles (Mullis et ah, supra). One skilled in the art understands that the one or more nucleotide mismatches that distinguish between the variant allele and other alleles are often located in the center of an allele-specific oligonucleotide primer to be used in the allele- specific oligonucleotide hybridization. In contrast, an allele-specific oligonucleotide primer to be used in PCR amplification generally contains the one or more nucleotide mismatches that distinguish between the variant and other alleles at the 3' end of the primer.

[0205] A heteroduplex mobility assay (HMA) is another well-known assay that can be used for genotyping in the methods of the present invention to determine the presence or absence of a particular variant allele or haplotype in the NOD2 gene or other genetic marker. HMA is useful for detecting the presence of a variant allele since a DNA duplex carrying a mismatch has reduced mobility in a polyacrylamide gel compared to the mobility of a perfectly base- paired duplex {see, Delwart et al, Science, 262: 1257-1261 (1993); White et ah, Genomics, 12:301 -306 (1992)).

[0206] The technique of single strand conformational polymorphism (SSCP) can also be useful for genotyping in the methods described herein to determine the presence or absence of a particular variant allele or haplotype in the NOD2 gene or other genetic marker (see, Hayashi, Methods Applic, 1 :34-38 (1991 )). This technique is used to detect variant alleles based on differences in the secondary structure of single-stranded DNA that produce an altered electrophoretic mobility upon non-denaturing gel electrophoresis. Variant alleles are detected by comparison of the electrophoretic pattern of the test fragment to corresponding standard fragments containing known alleles. [0207] Denaturing gradient gel electrophoresis (DGGE) can also be useful in the methods of the invention to determine the presence or absence of a particular variant allele or haplotype in the NOD2 gene or other genetic marker. In DGGE, double-stranded DNA is electrophoresed in a gel containing an increasing concentration of denaturant; double- stranded fragments made up of mismatched alleles have segments that melt more rapidly, causing such fragments to migrate differently as compared to perfectly complementary sequences (see, Sheffield et ai, "Identifying DNA Polymorphisms by Denaturing Gradient Gel Electrophoresis" in Innis et ai , supra, 1990).

[0208] In certain preferred embodiments, the presence or absence of one or more NOD2 variant alleles (e.g., SNP 8, SNP 12, and/or SNP 13) is determined using the NOD2/CARD15 assay available from Prometheus Laboratories Inc. (San Diego, CA; Cat. #6000).

[0209] Other molecular methods useful for genotyping an individual are known in the art and useful in the methods of the present invention. Such well-known genotyping approaches include, without limitation, automated sequencing and RNase mismatch techniques (see, Winter et ai, Proc. Natl. Acad. Set, 82:7575-7579 (1985)). Furthermore, one skilled in the art understands that, where the presence or absence of multiple variant alleles is to be determined, individual variant alleles can be detected by any combination of molecular methods. See, in general, Birren et al. (Eds.) Genome Analysis: A Laboratory Manual Volume 1 (Analyzing DNA) New York, Cold Spring Harbor Laboratory Press ( 1997). In addition, one skilled in the art understands that multiple variant alleles can be detected in individual reactions or in a single reaction (a "multiplex" assay).

[0210] In view of the above, one skilled in the art realizes that the methods of the present invention for predicting a patient's risk of developing postoperative complications following pouch surgery (e.g., by determining the presence or absence of one or more NOD2 variant alleles) can be practiced using one or any combination of the well-known genotyping assays described above or other assays known in the art.

VIII. miRNA Extraction, Purification, and Enrichment

[0211] For embodiments utilizing miRNA, cells are isolated and lysed to produce a cellular extract, small RNA species such as miRNAs may be extracted, purified, and/or enriched from the cellular extract by any technique known in the art. [0212] In some instances, an alcohol solution may be added to, mixed with, or incubated with the lysate or cellular extract prior to extraction of miRNAs. The alcohol solution may comprise at least one alcohol and typically ranges from about 5% to about 100% in the concentration of alcohol. In specific embodiments, the amount of alcohol solution added to the lysate renders it with an alcohol concentration of about 35% to about 70%, or about 50% to about 60%. In other specific embodiments, the amount of alcohol solution added to the lysate gives it an alcohol concentration of about 55%. Suitable alcohols include, but are not limited to, ethanol, propanol, isopropanol, methanol, and mixtures thereof. It is further contemplated that an alcohol solution may be used in additional steps in methods for precipitating RNA.

[0213] In certain aspects, miRNAs may be extracted from the lysate or cellular extract with an extraction solution comprising a non-alcohol organic solvent prior to applying the lysate or cellular extract to a solid support. In specific embodiments, the extraction solution contains a non-alcohol organic solvent such as phenol and/or chloroform. The non-alcohol organic solvent solution is understood to contain at least one non-alcohol organic solvent, though it may also contain an alcohol. The concentrations described above with respect to alcohol solutions are applicable to concentrations of solutions having non-alcohol organic solvents. In certain instances, equal amounts of the lysate and phenol and/or chloroform are mixed. In specific embodiments, the alcohol solution is added to the lysate before extraction with a non-alcohol organic solvent.

[0214] In some embodiments, extraction of miRNAs from the lysate or cellular extract includes using a solid support, such as a mineral or polymer support. A "solid support" includes a physical structure containing a material which contacts the lysate and that does not irreversibly react to macromolecules in the lysate, particularly with small RNA molecules such as miRNAs. In particular embodiments, the solid support binds small RNA molecules; in additional cases, it binds small RNA molecules, but does not bind one or more other types of macromolecules in the sample. The material in the solid support may include a mineral or polymer, in which case the support is referred to as a "mineral or polymer support." Mineral or polymer supports include supports involving silica. In some embodiments, the silica is glass. Suitable supports include, but are not limited to, beads, columns, and filters. In further embodiments, the mineral or polymer support is a glass fiber filter (GFF) or column. [0215] In certain other embodiments, the mineral or polymer support may include polymers or nonpolymers with electronegative groups. In some instances, the material comprises polyacrylate, polystyrene, latex, polyacrylonitrile, polyvinylchloride, methacrylate, and/or methyl methacrylate.

[0216] In further embodiments, a lysate that may or may not have been mixed with an alcohol or non-alcohol organic solvent solution is applied to a solid support and the RNA (containing miRNAs) is eluted from the support.

[0217] After a lysate is applied or mixed with a solid support, the material may be washed with a solution. In some embodiments, a mineral or polymer support is washed with a first wash solution after applying the lysate to the mineral or polymer support. In further embodiments, a wash solution comprises a chaotropic or reducing agent. The chaotropic agent is guanidinium in some wash solutions. A wash solution includes alcohol in some embodiments, and in some cases, it has both alcohol and guanidinium. It is further contemplated that the extraction step include 1 , 2, 3, 4, 5, or more washes with a wash solution. The wash solution used when more than one washing is involved may be the same or different. In some embodiments, the wash solutions have the same components, but in different concentrations from each other. It is generally understood that molecules that come through the material in a wash cycle are discarded.

[0218] The desired RNA molecules are typically eluted from the solid support. In certain embodiments, small RNA molecules {e.g., miRNAs) are eluted from a solid support such as a mineral or polymer support at a temperature of about 60°C to about 100°C. The temperature at which the RNA molecules are eluted may be about or at least about 5 to about 100°C or more, or any range therein. The molecules may be eluted with any elution solution. In some embodiments, the elution solution is an ionic solution. In particular embodiments, the elution solution includes up to about 10 mM salt {e.g., about 0.1 , 0.5, 1 , 5, 10, or more mM salt). In certain embodiments, the salt consists of a combination of Li⁺, Na⁺, K⁺, or NH₄ ⁺ as the cation and CI^", Br^", Γ, ethylenediaminetetraacetate, or citrate as the anion.

[0219] Additional steps include passing the small RNA molecules through a glass fiber filter (GFF) while binding only the larger RNAs. In some embodiments, the passed small RNA molecules are captured on a second GFF and then eluted. Material that is not captured on the second GFF filter may be discarded or not used. [0220] In a specific embodiment, the extraction of miRNAs is performed as follows:

adding an extraction solution to a cellular lysate containing miRNAs; adding an alcohol solution to the extracted sample; applying the sample to a mineral or polymer support; and eluting the RNA containing miRNAs from the mineral or polymer support with an ionic solution. In some embodiments, the eluted sample is enriched at least about 10-fold for miRNAs by mass.

[0221] As a non-limiting example, the extraction, purification, and enrichment of miRNAs may be performed according to the following protocol. 60 μΐ of 2M Na-acetate, pH 4.0, is added to a cellular lysate, followed immediately by 0.6 ml of acid phenol-chloroform. In certain instances, ethanol is added to the cellular lysate before phenol-chloroform extraction to provide a final concentration of about 55% ethanol. After 30 sec of vigorous agitation, the aqueous phase is separated by centrifugation at 16,000 x G for 5 min. Four 100 μΐ aliquots of this aqueous phase are used in four separate separations. The four aliquots have 100 μΐ of 40%, 50%, 60%, and 70% ethanol added to each, then are passed through glass fiber filters as in the RNAqueous procedure (Ambion, Inc.; Austin, TX). The 20%, 25%, 30%, and 35% ethanol solutions that passed through these filters (the flow-through) are then adjusted to 55% ethanol final concentration by the addition of 156, 133, 1 1 1 , and 88.9 μΐ of ethanol, respectively. All four samples are passed over separate glass fiber filter columns. The filters are then washed with 0.7 ml of 4 M guanidinium isocyanate (GuSCN)/70% ethanol, followed by two washes with 0.5 ml 80% alcohol/0.1 M NaCl/4.5 mM EDTA/10 mM TrisHCl, pH 7.5. After each wash is passed through the filter, the collection tube is emptied and replaced. Each wash is passed through the filter by centrifugation as per the RNAqueous protocol (Ambion, Inc.). The sample is then eluted off the filter with 100 μΐ of 0.1 mM EDTA, pH 8.0, which is applied directly to the filter at room temperature and centrifuged through into a fresh collection tube.

[0222] Additional methods for extracting, purifying, and enriching miRNAs are described in, e.g., U.S. Patent Publication No. 20050059024; and the ra Vana™ miRNA Isolation Kit Protocol (Ambion, Inc.; Austin, TX), the disclosures of which are herein incorporated by reference in their entirety for all purposes.

IX. Statistical Analysis

[0223] In some aspects, the present invention provides methods, systems, and code for predicting or determining a risk of developing dysplasia, cancer, and/or an inflammatory complication (e.g., pouchitis) in an individual following pouch surgery (e.g., IPAA). In particular embodiments, median analysis or quantile analysis is applied to the presence, level, and/or genotype of one or more markers determined by any of the assays described herein to predict a patient's risk of developing postoperative complications following pouch surgery. In certain other embodiments, one or more learning statistical classifier systems are applied to the presence, level, and/or genotype of one or more markers determined by any of the assays described herein. The statistical analyses of the present invention advantageously provide improved sensitivity, specificity, negative predictive value, positive predictive value, and/or overall accuracy for predicting or determining a risk of developing dysplasia, cancer, and/or an inflammatory complication (e.g., pouchitis) in an individual following pouch surgery (e.g., IPAA).

[0224] The term "statistical analysis" or "statistical algorithm" or "statistical process" includes any of a variety of statistical methods and models used to determine relationships between variables. In the present invention, the variables are the presence, level, or genotype of at least one marker of interest. Any number of markers can be analyzed using a statistical analysis described herein. For example, the presence, level, or genotype of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more markers can be included in a statistical analysis. In one embodiment, logistic regression is used. In another embodiment, linear regression is used. In some embodiments, the statistical analyses of the present invention comprise a median analysis of one or more markers, e.g., whether a marker level is above or below a reference value such as a median concentration level. In certain preferred embodiments, the statistical analyses of the present invention comprise a quantile measurement of one or more markers, e.g., within a given population, as a variable. Quantiles are a set of "cut points" that divide a sample of data into groups containing (as far as possible) equal numbers of observations. For example, quartiles are values that divide a sample of data into four groups containing (as far as possible) equal numbers of observations. The lower quartile is the data value a quarter way up through the ordered data set; the upper quartile is the data value a quarter way down through the ordered data set. Quintiles are values that divide a sample of data into five groups containing (as far as possible) equal numbers of observations. The present invention can also include the use of percentile ranges of marker levels (e.g., tertiles, quartile, quintiles, etc.), or their cumulative indices (e.g., quartile sums of marker levels to obtain quartile sum scores (QSS), etc.) as variables in the statistical analyses (just as with continuous variables). [0225] In preferred embodiments, the present invention involves detecting or determining the presence, level (e.g., magnitude), and/or genotype of one or more markers of interest using quartile analysis. In this type of statistical analysis, the level of a marker of interest is defined as being in the first quartile (<25%), second quartile (25-50%), third quartile (51 %- <75%), or fourth quartile (75- 100%) in relation to a reference database of samples. These quartiles may be assigned a quartile score of 1 , 2, 3, and 4, respectively. In certain instances, a marker that is not detected in a sample is assigned a quartile score of 0 or 1 , while a marker that is detected (e.g., present) in a sample (e.g., sample is positive for the marker) is assigned a quartile score of 4. In some embodiments, quartile 1 represents samples with the lowest marker levels, while quartile 4 represent samples with the highest marker levels. In other embodiments, quartile 1 represents samples with a particular marker genotype (e.g., wild- type allele), while quartile 4 represent samples with another particular marker genotype (e.g., allelic variant). The reference database of samples can include a large spectrum of IBD (e.g., CD and/or UC) patients before and/or after pouch surgery. From such a database, quartile cut-offs can be established. A non-limiting example of quartile analysis suitable for use in the present invention is described in, e.g., Mow et ah , Gastroenterology, 126:414-24 (2004).

[0226] In some embodiments, the statistical analyses of the present invention comprise one or more learning statistical classifier systems. As used herein, the term "learning statistical classifier system" includes a machine learning algorithmic technique capable of adapting to complex data sets (e.g., panel of markers of interest) and making decisions based upon such data sets. In some embodiments, a single learning statistical classifier system such as a decision/classification tree (e.g., random forest (RF) or classification and regression tree (C&RT)) is used. In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem. Examples of learning statistical classifier systems include, but are not limited to, those using inductive learning (e.g., decision/classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connectionist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feedforward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g., passive learning in a known environment such as na^'ive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programming. Other learning statistical classifier systems include support vector machines (e.g., Kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithms, Gauss-Newton algorithms, mixtures of Gaussians, gradient descent algorithms, and learning vector quantization (LVQ).

[0227] Random forests are learning statistical classifier systems that are constructed using an algorithm developed by Leo Breiman and Adele Cutler. Random forests use a large number of individual decision trees and decide the class by choosing the mode (i.e., most frequently occurring) of the classes as determined by the individual trees. Random forest analysis can be performed, e.g., using the RandomForests software available from Salford Systems (San Diego, CA). See, e.g., Breiman, Machine Learning, 45:5-32 (2001 ); and http://stat-www.berkeley.edu/users/breiman/RandomForests/cc_home.htm, for a description of random forests.

[0228] Classification and regression trees represent a computer intensive alternative to fitting classical regression models and are typically used to determine the best possible model for a categorical or continuous response of interest based upon one or more predictors.

Classification and regression tree analysis can be performed, e.g., using the C&RT software available from Salford Systems or the Statistica data analysis software available from

StatSoft, Inc. (Tulsa, OK). A description of classification and regression trees is found, e.g., in Breiman et al. "Classification and Regression Trees," Chapman and Hall, New York (1984); and Steinberg et al , "CART: Tree-Structured Non-Parametric Data Analysis," Salford Systems, San Diego, ( 1995).

[0229] Neural networks are interconnected groups of artificial neurons that use a mathematical or computational model for information processing based on a connectionist approach to computation. Typically, neural networks are adaptive systems that change their structure based on external or internal information that flows through the network. Specific examples of neural networks include feed-forward neural networks such as perceptrons, single-layer perceptrons, multi-layer perceptrons, backpropagation networks, AD ALINE networks, MAD ALINE networks, Learnmatrix networks, radial basis function (RBF) networks, and self-organizing maps or Kohonen self-organizing networks; recurrent neural networks such as simple recurrent networks and Hopfield networks; stochastic neural networks such as Boltzmann machines; modular neural networks such as committee of machines and associative neural networks; and other types of networks such as

instantaneously trained neural networks, spiking neural networks, dynamic neural networks, and cascading neural networks. Neural network analysis can be performed, e.g., using the Statistica data analysis software available from StatSoft, Inc. See, e.g., Freeman et al., In "Neural Networks: Algorithms, Applications and Programming Techniques," Addison- Wesley Publishing Company (1991); Zadeh, Information and Control, 8:338-353 ( 1965); Zadeh, "IEEE Trans, on Systems, Man and Cybernetics," 3:28-44 (1973); Gersho et al., In "Vector Quantization and Signal Compression," Kluywer Academic Publishers, Boston, Dordrecht, London (1992); and Hassoun, "Fundamentals of Artificial Neural Networks," MIT Press, Cambridge, Massachusetts, London ( 1995), for a description of neural networks.

[0230] Support vector machines are a set of related supervised learning techniques used for classification and regression and are described, e.g., in Cristianini et al. , "An Introduction to Support Vector Machines and Other Kernel-Based Learning Methods," Cambridge

University Press (2000). Support vector machine analysis can be performed, e.g., using the SVM^" software developed by Thorsten Joachims (Cornell University) or using the

LIBSVM software developed by Chih-Chung Chang and Chih-Jen Lin (National Taiwan University).

[0231] The various statistical methods described herein can be trained and tested using a cohort of samples {e.g., serological and/or genomic samples) from healthy individuals and IBD {e.g., CD and/or UC) patients before and/or after pouch surgery. For example, samples from patients diagnosed by a physician, and preferably by a gastroenterologist, as having IBD or a clinical subtype thereof using a biopsy, colonoscopy, or an immunoassay as described in, e.g., U.S. Patent No. 6,218,129, are suitable for use in training and testing the statistical methods of the present invention. Samples from patients diagnosed with IBD can also be stratified into Crohn's disease or ulcerative colitis using an immunoassay as described in, e.g., U.S. Patent Nos. 5,750,355 and 5,830,675. Samples from healthy individuals can include those that were not identified as IBD samples. One skilled in the art will know of additional techniques and diagnostic criteria for obtaining a cohort of patient samples that can be used in training and testing the statistical methods of the present invention.

[0232] As used herein, the term "sensitivity" refers to the probability that a prognostic or predictive method, system, or code of the present invention gives a positive result when the sample is positive, e.g., having the predicted prognostic outcome or response to therapy. Sensitivity is calculated as the number of true positive results divided by the sum of the true positives and false negatives. Sensitivity essentially is a measure of how well the present invention correctly identifies those who have the predicted prognostic outcome or response to therapy from those who do not have the predicted prognosis or response. The statistical methods and models can be selected such that the sensitivity is at least about 60%, and can be, e.g., at least about 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0233] The term "specificity" refers to the probability that a prognostic or predictive method, system, or code of the present invention gives a negative result when the sample is not positive, e.g., not having the predicted prognostic outcome or response to therapy.

Specificity is calculated as the number of true negative results divided by the sum of the true negatives and false positives. Specificity essentially is a measure of how well the present invention excludes those who do not have the predicted prognostic outcome or response to therapy from those who do have the predicted prognosis or response. The statistical methods and models can be selected such that the specificity is at least about 60%, and can be, e.g., at least about 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0234] As used herein, the term "negative predictive value" or "NPV" refers to the probability that an individual identified as not having the predicted prognostic outcome or response to therapy actually does not have the predicted prognosis or therapeutic response. Negative predictive value can be calculated as the number of true negatives divided by the sum of the true negatives and false negatives. Negative predictive value is determined by the characteristics of the prognostic method, system, or code as well as the prevalence of the disease in the population analyzed. The statistical methods and models can be selected such that the negative predictive value in a population having a disease prevalence is in the range of about 70% to about 99% and can be, for example, at least about 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0235] The term "positive predictive value" or "PPV" refers to the probability that an individual identified as having the predicted prognostic outcome or response to therapy actually has the predicted prognosis or therapeutic response. Positive predictive value can be calculated as the number of true positives divided by the sum of the true positives and false positives. Positive predictive value is determined by the characteristics of the prognostic method, system, or code as well as the prevalence of the disease in the population analyzed. The statistical methods and models can be selected such that the positive predictive value in a population having a disease prevalence is in the range of about 70% to about 99% and can be, for example, at least about 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0236] Predictive values, including negative and positive predictive values, are influenced by the prevalence of the disease in the population analyzed. In some instances, the statistical methods and models can be selected to produce a desired clinical parameter for a clinical population with a particular IBD prevalence. For example, statistical methods and models can be selected for an IBD prevalence of up to about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, which can be seen, e.g., in a clinician's office such as a gastroenterologist's office or a general

practitioner's office.

[0237] As used herein, the term "overall agreement" or "overall accuracy" refers to the accuracy with which a method, system, or code of the present invention predicts prognostic outcome or response to therapy. Overall accuracy is calculated as the sum of the true positives and true negatives divided by the total number of sample results and is affected by the prevalence of the disease in the population analyzed. For example, the statistical methods and models can be selected such that the overall accuracy in a patient population having a disease prevalence is at least about 40%, and can be, e.g., at least about 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

X. Disease Classification System

[0238] Figure 1 illustrates a disease classification system (DCS) (100) according to one embodiment of the present invention. As shown therein, a DCS includes a DCS intelligence module (105), such as a computer, having a processor (115) and memory module (110). The intelligence module also includes communication modules (not shown) for transmitting and receiving information over one or more direct connections (e.g., USB, Firewire, or other interface) and one or more network connections (e.g. , including a modem or other network interface device). The memory module may include internal memory devices and one or more external memory devices. The intelligence module also includes a display module (125), such as a monitor or printer. In one aspect, the intelligence module receives data such as patient test results from a data acquisition module such as a test system (150), either through a direct connection or over a network (140). For example, the test system may be configured to run multianalyte tests on one or more patient samples (155) and automatically provide the test results to the intelligence module. The data may also be provided to the intelligence module via direct input by a user or it may be downloaded from a portable medium such as a compact disk (CD) or a digital versatile disk (DVD). The test system may be integrated with the intelligence module, directly coupled to the intelligence module, or it may be remotely coupled with the intelligence module over the network. The intelligence module may also communicate data to and from one or more client systems (130) over the network as is well known. For example, a requesting physician or healthcare provider may obtain and view a report from the intelligence module, which may be resident in a laboratory or hospital, using a client system (130).

[0239] The network can be a LAN (local area network), WAN (wide area network), wireless network, point-to-point network, star network, token ring network, hub network, or other configuration. As the most common type of network in current use is a TCP/IP (Transfer Control Protocol and Internet Protocol) network such as the global internetwork of networks often referred to as the "Internet" with a capital "I," that will be used in many of the examples herein, but it should be understood that the networks that the present invention might use are not so limited, although TCP/IP is the currently preferred protocol.

[0240] Several elements in the system shown in Figure 1 may include conventional, well- known elements that need not be explained in detail here. For example, the intelligence module could be implemented as a desktop personal computer, workstation, mainframe, laptop, etc. Each client system could include a desktop personal computer, workstation, laptop, PDA, cell phone, or any WAP-enabled device or any other computing device capable of interfacing directly or indirectly to the Internet or other network connection. A client system typically runs an HTTP client, e.g., a browsing program, such as Microsoft's Internet

Explorer browser, Netscape's Navigator browser, Opera's browser, or a WAP-enabled browser in the case of a cell phone, PDA or other wireless device, or the like, allowing a user of the client system to access, process, and view information and pages available to it from the intelligence module over the network. Each client system also typically includes one or more user interface devices, such as a keyboard, a mouse, touch screen, pen or the like, for interacting with a graphical user interface (GUI) provided by the browser on a display (e.g., monitor screen, LCD display, etc. ) (135) in conjunction with pages, forms, and other information provided by the intelligence module. As discussed above, the present invention is suitable for use with the Internet, which refers to a specific global internetwork of networks. However, it should be understood that other networks can be used instead of the Internet, such as an intranet, an extranet, a virtual private network (VPN), a non-TCP/IP based network, any LAN or WAN, or the like.

[0241] According to one embodiment, each client system and all of its components are operator configurable using applications, such as a browser, including computer code run using a central processing unit such as an Intel^® Pentium^® processor or the like. Similarly, the intelligence module and all of its components might be operator configurable using application(s) including computer code run using a central processing unit (115) such as an Intel Pentium processor or the like, or multiple processor units. Computer code for operating and configuring the intelligence module to process data and test results as described herein is preferably downloaded and stored on a hard disk, but the entire program code, or portions thereof, may also be stored in any other volatile or non-volatile memory medium or device as is well known, such as a ROM or RAM, or provided on any other computer readable medium (160) capable of storing program code, such as a compact disk (CD) medium, digital versatile disk (DVD) medium, a floppy disk, ROM, RAM, and the like.

[0242] The computer code for implementing various aspects and embodiments of the present invention can be implemented in any programming language that can be executed on a computer system such as, for example, in C, C++, C#, HTML, Java, JavaScript, or any other scripting language, such as VBScript. Additionally, the entire program code, or portions thereof, may be embodied as a carrier signal, which may be transmitted and downloaded from a software source (e.g., server) over the Internet, or over any other conventional network connection as is well known (e.g., extranet, VPN, LAN, etc.) using any communication medium and protocols (e.g., TCP/I P, HTTP, HTTPS, Ethernet, etc.) as are well known.

[0243] According to one embodiment, the intelligence module implements a disease classification process for analyzing patient test results to determine a prognostic outcome or response to therapy. The data may be stored in one or more data tables or other logical data structures in memory (110) or in a separate storage or database system coupled with the intelligence module. One or more statistical analyses or processes are typically applied to a data set including test data for a particular patient. For example, the test data might include a marker profile, which comprises data indicating the presence, level, and/or genotype of at least one marker in a sample from the patient. In one embodiment, a statistical analysis such as a quantile (e.g., quartile) analysis is applied to test data for a particular patient, wherein the test data comprises the presence, level, and/or genotype of at least one marker determined in a sample from the patient. The statistically derived decision(s) may be displayed on a display device associated with or coupled to the intelligence module, or the decision(s) may be provided to and displayed at a separate system, e.g., a client system (130). In particular embodiments, the statistically derived decision(s) may be displayed in the form of a report or print-out, which can optionally include a look-up table, chart, graph, or model to enable a physician to compare and interpret the displayed results to make a reasoned prognosis or therapeutic response prediction.

XL Therapy and Therapeutic Monitoring

[0244] Once the risk of developing postoperative complications to pouch surgery has been predicted or the response to therapy of an individual having such postoperative complications has been predicted according to the methods described herein, the present invention may further comprise recommending a course of therapy based upon the prediction. In certain instances, the present invention may further comprise administering to the individual a therapeutically effective amount of a therapeutic agent such as an IBD therapeutic agent for gastrointestinal-related inflammatory complications and/or an anticancer drug for dysplasia- and cancer-related complications. For therapeutic applications, the IBD therapeutic agent and/or an anticancer drug can be administered alone or co-administered in combination with one or more additional therapeutic agents and/or one or more drugs that reduce the side- effects associated with the therapeutic agent. Examples of IBD therapeutic agents include, but are not limited to, biologic agents, conventional drugs, and combinations thereof.

Examples of anticancer drugs include, but are not limited to, anti-signaling agents (i.e., cytostatic drugs) such as monoclonal antibodies and tyrosine kinase inhibitors; antiproliferative agents; chemotherapeutic agents (i.e., cytotoxic drugs); hormonal therapeutic agents; radiotherapeutic agents; vaccines; and/or any other compound with the ability to reduce or abrogate the uncontrolled growth of aberrant cells such as cancerous cells. As such, the present invention advantageously enables a clinician to practice "personalized medicine" by guiding treatment decisions and informing therapy selection for post-surgery complications such that the right drug is given to the right patient at the right time.

[0245] Therapeutic agents can be administered with a suitable pharmaceutical excipient as necessary and can be carried out via any of the accepted modes of administration. Thus, administration can be, for example, intravenous, topical, subcutaneous, transcutaneous, transdermal, intramuscular, oral, buccal, sublingual, gingival, palatal, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, or by inhalation. By "co-administer" it is meant that a therapeutic agent is administered at the same time, just prior to, or just after the administration of a second drug (e.g., another therapeutic agent, a drug useful for reducing the side-effects of the therapeutic agent, etc.).

[0246] A therapeutically effective amount of a therapeutic agent may be administered repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, or more times, or the dose may be administered by continuous infusion. The dose may take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.

[0247] As used herein, the term "unit dosage form" includes physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of a therapeutic agent calculated to produce the desired onset, tolerability, and/or therapeutic effects, in association with a suitable pharmaceutical excipient (e.g., an ampoule). In addition, more concentrated dosage forms may be prepared, from which the more dilute unit dosage forms may then be produced. The more concentrated dosage forms thus will contain substantially more than, e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times the amount of the therapeutic agent.

[0248] Methods for preparing such dosage forms are known to those skilled in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co., Easton, PA ( 1990)) . The dosage forms typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, diluents, tissue permeation enhancers, solubilizers, and the like. Appropriate excipients can be tailored to the particular dosage form and route of administration by methods well known in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra).

[0249] Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols, e.g., Carbopol 941 , Carbopol 980, Carbopol 981 , etc. The dosage forms can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preserving agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates {i.e., the parabens); pH adjusting agents such as inorganic and organic acids and bases; sweetening agents; and flavoring agents. The dosage forms may also comprise biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

[0250] For oral administration, the therapeutically effective dose can be in the form of tablets, capsules, emulsions, suspensions, solutions, syrups, sprays, lozenges, powders, and sustained-release formulations. Suitable excipients for oral administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.

[0251] In some embodiments, the therapeutically effective dose takes the form of a pill, tablet, or capsule, and thus, the dosage form can contain, along with a therapeutic agent, any of the following: a diluent such as lactose, sucrose, dicalcium phosphate, and the like; a disintegrant such as starch or derivatives thereof; a lubricant such as magnesium stearate and the like; and a binder such a starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof. A therapeutic agent can also be formulated into a suppository disposed, for example, in a polyethylene glycol (PEG) carrier.

[0252] Liquid dosage forms can be prepared by dissolving or dispersing a therapeutic agent and optionally one or more pharmaceutically acceptable adjuvants in a carrier such as, for example, aqueous saline (e.g., 0.9% w/v sodium chloride), aqueous dextrose, glycerol, ethanol, and the like, to form a solution or suspension, e.g., for oral, topical, or intravenous administration. A therapeutic agent can also be formulated into a retention enema.

[0253] For topical administration, the therapeutically effective dose can be in the form of emulsions, lotions, gels, foams, creams, jellies, solutions, suspensions, ointments, and transdermal patches. For administration by inhalation, a therapeutic agent can be delivered as a dry powder or in liquid form via a nebulizer. For parenteral administration, the

therapeutically effective dose can be in the form of sterile injectable solutions and sterile packaged powders. Preferably, injectable solutions are formulated at a pH of from about 4.5 to about 7.5.

[0254] The therapeutically effective dose can also be provided in a lyophilized form. Such dosage forms may include a buffer, e.g., bicarbonate, for reconstitution prior to

administration, or the buffer may be included in the lyophilized dosage form for

reconstitution with, e.g., water. The lyophilized dosage form may further comprise a suitable vasoconstrictor, e.g., epinephrine. The lyophilized dosage form can be provided in a syringe, optionally packaged in combination with the buffer for reconstitution, such that the reconstituted dosage form can be immediately administered to an individual.

[0255] In therapeutic use for the treatment of complications associated with pouch surgery, a therapeutic agent can be administered at the initial dosage of from about 0.001 mg/kg to about 1000 mg/kg daily. A daily dose range of from about 0.01 mg/kg to about 500 mg/kg, from about 0.1 mg/kg to about 200 mg/kg, from about 1 mg/kg to about 100 mg/kg, or from about 10 mg/kg to about 50 mg/kg, can be used. The dosages, however, may be varied depending upon the requirements of the individual, the severity of symptoms, and the therapeutic agent being employed. For example, dosages can be empirically determined considering the type and severity of symptoms in an individual. The dose administered to an individual, in the context of the present invention, should be sufficient to affect a beneficial therapeutic response in the individual over time. The size of the dose can also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular therapeutic agent in an individual. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the therapeutic agent. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.

[0256] As used herein, the term "IBD therapeutic agent" includes all pharmaceutically acceptable forms of a drug that is useful for treating one or more symptoms associated with IBD or one or more gastrointestinal-related inflammatory complications associated with pouch surgery. For example, the IBD therapeutic agent can be in a racemic or isomeric mixture, a solid complex bound to an ion exchange resin, or the like. In addition, the IBD therapeutic agent can be in a solvated form. The term is also intended to include all pharmaceutically acceptable salts, derivatives, and analogs of the IBD therapeutic agent being described, as well as combinations thereof. For example, the pharmaceutically acceptable salts of an IBD therapeutic agent include, without limitation, the tartrate, succinate, tartarate, bitartarate, dihydrochloride, salicylate, hemisuccinate, citrate, maleate, hydrochloride, carbamate, sulfate, nitrate, and benzoate salt forms thereof, as well as combinations thereof and the like. Any form of an IBD therapeutic agent is suitable for use in the methods of the present invention, e.g., a pharmaceutically acceptable salt of an IBD therapeutic agent, a free base of an IBD therapeutic agent, or a mixture thereof. Examples of suitable IBD therapeutic agents include, but are not limited to, biologic agents, conventional drugs, and combinations thereof.

[0257] Biologic agents include, e.g., anti-cytokine and chemokine antibodies such as antitumor necrosis factor alpha (TNFa) antibodies. Non-limiting examples of anti-TNFa antibodies include: chimeric monoclonal antibodies such as infliximab (Remicade^®) (Centocor, Inc.; Horsham, PA), which is a chimeric IgG l anti-TNFa monoclonal antibody; humanized monoclonal antibodies such as CDP571 and the PEGylated CDP870; fully human monoclonal antibodies such as adalimumab (Humira^®) (Abbott Laboratories; Abbott Park, IL); p75 fusion proteins such as etanercept (Enbrel^®) (Amgen; Thousand Oaks, CA; Wyeth Pharmaceuticals Inc.; Collegeville, PA), small molecules (e.g., MAP kinase inhibitors); and combinations thereof. See, Ghosh, Novartis Found Symp., 263: 193-205 (2004).

[0258] Other biologic agents include, e.g., anti-cell adhesion antibodies such as natalizumab (Tysabri^®) (Elan Pharmaceuticals, Inc.; Dublin, Ireland; Biogen Idee;

Cambridge, MA), which is a humanized monoclonal antibody against the cellular adhesion molecule a4-integrin, and MLN-02 (Millennium Pharmaceuticals; Cambridge, MA), which is a humanized IgGl anti-a4p7-integrin monoclonal antibody; anti-T cell agents; anti-CD3 antibodies such as visilizumab (Nuvion^®) (PDL BioPharma; Incline Village, NV), which is a humanized IgG2M3 anti-CD3 onoclonal antibody; anti-CD4 antibodies such as priliximab (cM-T412) (Centocor, Inc.; Horsham, PA), which is a chimeric anti-CD4 monoclonal antibody; anti-IL-2 receptor alpha (CD25) antibodies such as daclizumab Zenapax^®) (PDL BioPharma; Incline Village, NV; Roche; Nutley, NJ), which is a humanized IgGl anti-CD25 monoclonal antibody, and basiliximab (Simulect^®) (Novartis; Basel, Switzerland), which is a chimeric IgGl anti-CD25 monoclonal antibody; and combinations thereof.

[0259] In addition to the foregoing biological agents, the miRs of Table 2, or an inhibitor of the miRs of Table 2 are useful in the present invention. As such, in certain embodiments, the present invention provides treatment or prevention of inflammatory complications associated with pouch surgery by introducing into or providing to a patient in need thereof an effective amount of i) an miRNA inhibitor molecule or ii) a miRNA molecule that corresponds to an miRNA sequence set forth in Table 2.

[0260] One useful formulation for the delivery of miRs are liposomes. Liposomes and emulsions are well-known examples of delivery vehicles that may be used to deliver nucleic acids of the invention. A nucleic acid of the invention can be administered in combination with a carrier or lipid to increase cellular uptake. For example, the oligonucleotide may be administered in combination with a cationic lipid. Examples of cationic lipids include, but are not limited to, lipofectin, DOTMA, DOPE, and DOTAP. The publication of

WO0071096, which is specifically incorporated by reference, describes different

formulations, such as a DOTAPrcholesterol or cholesterol derivative formulation that can effectively be used for gene therapy. Other disclosures also discuss different lipid or liposomal formulations including nanoparticles and methods of administration; these include, but are not limited to, U.S. Patent Publication 20030203865, 20020150626, 20030032615, and 20040048787, which are specifically incorporated by reference to the extent they disclose formulations and other related aspects of administration and delivery of nucleic acids. Methods used for forming particles are also disclosed in U.S. Pat. Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835, 5,972,901 , 6,200,801 , and 5,972,900, which are incorporated by reference for those aspects. The nucleic acids may also be administered in combination with a cationic amine such as poly (L-lysine).

[0261] Examples of conventional drugs include, without limitation, aminosalicylates (e.g., mesalazine, sulfasalazine, and the like), corticosteroids (e.g., prednisone), thiopurines (e.g., azathioprine, 6-mercaptopurine, and the like), methotrexate, free bases thereof,

pharmaceutically acceptable salts thereof, derivatives thereof, analogs thereof, and combinations thereof.

[0262] One skilled in the art will know of additional IBD therapeutic agents suitable for use in the present invention (see, e.g., Sands, Surg. Clin. North Am. , 86: 1045- 1064 (2006); Danese et al , Mini Rev. Med. Chem., 6:771 -784 (2006); Domenech, Digestion, 73 (Suppl. l ):67-76 (2006); Nakamura et al , World J. Gastroenterol , 12:4628-4635 (2006); and Gionchetti et al, World J. Gastroenterol , 12:3306-3313 (2006)).

[0263] In certain embodiments, the anticancer drug comprises an anti-signaling agent {i.e., a cytostatic drug) such as a monoclonal antibody or a tyrosine kinase inhibitor; an antiproliferative agent; a chemotherapeutic agent (i.e., a cytotoxic drug); a hormonal therapeutic agent; a radiotherapeutic agent; a vaccine; and/or any other compound with the ability to reduce or abrogate the uncontrolled growth of aberrant cells such as cancerous cells.

[0264] Examples of anti-signaling agents suitable for use in the present invention include, without limitation, monoclonal antibodies such as trastuzumab (Herceptin^®), alemtuzumab (Campath^®), bevacizumab (Avastin^®), cetuximab (Erbitux^®), gemtuzumab (Mylotarg^®), panitumumab (Vectibix™), rituximab (Rituxan^®), and tositumomab (BEXXAR^®); tyrosine kinase inhibitors such as gefitinib (Iressa^®), sunitinib (Sutent^®), erlotinib (Tarceva^®), lapatinib (GW-572016; Tykerb^®), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006; Nexavar^®), imatinib mesylate (Gleevec^®), leflunomide (SU101 ), and vandetanib (ZACTIMA™; ZD6474); and combinations thereof.

[0265] Exemplary anti-proliferative agents include mTOR inhibitors such as sirolimus (rapamycin), temsirolimus (CCI-779), and everolimus (RAD001); Akt inhibitors such as lL6-hydroxymethyl-chiro-inositol-2-(R)-2-0-methyl-3-0-octadecyl-s7i-glycerocarbonate, 9- methoxy-2-methylellipticinium acetate, 1 ,3-dihydro- 1 -( 1 -((4-(6-phenyl- 1 H-imidazo[4,5- g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one, 10-(4'-(N- diethylamino)butyl)-2-chlorophenoxazine, 3-formylchromone thiosemicarbazone (Cu(II)Cl₂ complex), API-2, a 15-mer peptide derived from amino acids 10-24 of the proto-oncogene TCL1 (Hiromura et al , J. Biol. Chem., 279:53407-53418 (2004), KP372-1 , and the compounds described in Kozikowski et al , J. Am. Chem. Soc , 125: 1 144-1 145 (2003) and Kau et al, Cancer Cell, 4:463-476 (2003); and combinations thereof.

[0266] Non-limiting examples of chemotherapeutic agents include platinum-based drugs (e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin, iproplatin, satraplatin, etc.), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, etc.), anti-metabolites (e.g., 5- fluorouracil, azathioprine, 6-mercaptopurine, methotrexate, leucovorin, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine (Gemzar^®), pemetrexed (ALIMTA^®), raltitrexed, etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel (Taxol^®), docetaxel (Taxotere^®), etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine, etoposide (VP16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.), pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and combinations thereof.

[0267] Examples of hormonal therapeutic agents include, without limitation, aromatase inhibitors (e.g., aminoglutethimide, anastrozole (Arimidex^®), letrozole (Femara^®), vorozole, exemestane (Aromasin^®), 4-androstene-3,6,17-trione (6-OXO), l ,4,6-androstatrien-3, 17- dione (ATD), formestane (Lentaron^®), etc.), selective estrogen receptor modulators (e.g., bazedoxifene, clomifene, fulvestrant, lasofoxifene, raloxifene, tamoxifen, toremifene, etc.), steroids (e.g., dexamethasone), finasteride, and gonadotropin-releasing hormone agonists (GnRH) such as goserelin, pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and combinations thereof.

[0268] Non-limiting examples of cancer vaccines useful in the present invention include ANYARA from Active Biotech, DCVax-LB from Northwest Biotherapeutics, EP-2101 from IDM Pharma, GV1001 from Pharmexa, IO-2055 from Idera Pharmaceuticals, INGN 225 from Introgen Therapeutics and Stimuvax from Biomira/Merck.

[0269] Examples of radiotherapeutic agents include, but are not limited to, radionuclides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ^,05Rh, ^{u l}Ag, ^{n ,} In, ^{1 17m}Sn, ¹⁴⁹Pm, ¹⁵³Sm, ^,66Ho, ^{l 77}Lu, ^{1 86}Re, ¹⁸⁸Re, ^{2l l}At, and ²¹²Bi, optionally conjugated to antibodies directed against tumor antigens.

[0270] An individual can also be monitored at periodic time intervals to assess the efficacy of a certain therapeutic regimen once prognostic and/or predictive information has been obtained from the individual's sample. For example, the presence or level of certain markers may change based on the therapeutic effect of a treatment such as a therapeutic drug. In certain embodiments, the patient can be monitored to assess response and understand the effects of certain drugs or treatments in an individualized approach. Additionally, patients may not respond to a drug, but the markers may change, suggesting that these patients belong to a special population (not responsive) that can be identified by their marker levels. These patients can be discontinued on their current therapy and alternative treatments prescribed. XII. Examples

[0271] The following examples are offered to illustrate, but not to limit, the claimed invention.

Example 1. Determination of ANCA Levels.

[0272] This example illustrates an analysis of ANCA levels in a sample using an ELISA assay.

[0273] A fixed neutrophil enzyme-linked immunosorbent assay (ELISA) may be used to detect ANCA as described in Saxon et al, J. Allergy Clin. Immunol, 86:202-210 (1990). Briefly, microtiter plates are coated with 2.5 x 10⁵ neutrophils per well from peripheral human blood purified by Ficoll-hypaque centrifugation and treated with 100% methanol for 10 minutes to fix the cells. Cells are incubated with 0.25% bovine serum albumin (BSA) in phosphate-buffered saline to block nonspecific antibody binding for 60 minutes at room temperature in a humidified chamber. Next, control and coded sera are added at a 1 : 100 dilution to the bovine serum/phosphate-buffered saline blocking buffer and incubated for 60 minutes at room temperature in a humidified chamber. Alkaline phosphatase-conjugated goat F(ab')₂ anti-human immunoglobulin G antibody (γ-chain specific; Jackson Immunoresearch Labs, Inc.; West Grove, Pa.) is added at a 1 : 1000 dilution to label neutrophil-bound antibody and incubated for 60 minutes at room temperature. A solution of p-nitrophenol phosphate substrate is added, and color development is allowed to proceed until absorbance at 405 nm in the positive control wells is 0.8- 1 .0 optical density units greater than the absorbance in blank wells.

[0274] ANCA levels may be determined relative to a standard consisting of pooled sera obtained from well-characterized pANCA-positive ulcerative colitis (UC) patients. Results are expressed as ELISA units. Sera with circulating ANCA levels exceeding the reference range value may also be termed ANCA positive, whereas numerical values that are below the reference range may also be termed ANCA negative.

Example 2. Determination of the Presence of pANCA.

[0275] This example illustrates an analysis of the presence or absence of pANCA in a sample using an immunofluorescence assay as described, e.g., in U.S. Patent Nos. 5,750,355 and 5,830,675. In particular, the presence of pANCA is detected by assaying for the loss of a positive value (e.g., loss of a detectable antibody marker and/or a specific cellular staining pattern as compared to a control) upon treatment of neutrophils with DNase.

[0276] Neutrophils isolated from a sample such as serum are immobilized on a glass side according to the following protocol:

1. Resuspend neutrophils in a sufficient volume of I X Hanks' Balanced Salt Solution (HBSS) to achieve about 2.5 x 10⁶ cells per ml.

2. Use a Cytospin3 centrifuge (Shandon, Inc.; Pittsburgh, PA) at 500 rpm for 5 minutes to apply 0.01 ml of the resuspended neutrophils to each slide.

3. Fix neutrophils to slide by incubating slides for 10 minutes in sufficient volume of 100% methanol to cover sample. Allow to air dry. The slides may be stored at -20°C.

[0277] The immobilized, fixed neutrophils are then treated with DNase as follows:

1. Prepare a DNase solution by combining 3 units of Promega RQ 1™ DNase (Promega;

Madison, WI) per ml buffer containing 40 mM of TRIS-HCl (pH 7.9), 10 mM of sodium chloride, 6 mM magnesium chloride, and 10 mM calcium chloride.

2. Rinse slides prepared using the above protocol with about 100 ml phosphate buffered saline (pH 7.0-7.4) for 5 minutes. Incubate immobilized neutrophils in 0.05 ml of DNase solution per slide for about 30 minutes at 37°C. Wash the slides three times with about 100-250 ml phosphate buffered saline at room temperature. The DNase reaction carried out as described herein causes substantially complete digestion of cellular DNA without significantly altering nuclear or cellular neutrophil morphology.

[0278] Next, an immunofluorescence assay is performed on the DNase-treated, fixed neutrophils according to the following protocol:

1. Add 0.05 ml of a 1 :20 dilution of human sera in phosphate buffered saline to slides treated with DNase and to untreated slides. Add 0.05 ml phosphate buffered saline to clean slides as blanks. Incubate for about 0.5 to 1.0 hour at room temperature in sufficient humidity to minimize volume loss.

2. Rinse off sera by dipping into a container having 100-250 ml phosphate buffered saline.

3. Soak slide in phosphate buffered saline for 5 minutes. Blot lightly.

4. Add 0.05 ml goat F(ab')₂ anti-human IgG^)-FITC (Tago Immunologicals;

Burlingame, CA), at a 1 : 1000 antibody:phosphate buffered saline dilution, to each slide. Incubate for 30 minutes at room temperature in sufficient humidity to minimize volume loss.

5. Rinse off antibody with 100-250 ml phosphate buffered saline. Soak slides for 5

minutes in 100-250 ml phosphate buffered saline, then allow to air dry.

6. Read fluorescence pattern on fluorescence microscope at 40X.

7. If desired, any DNA can be stained with propidium iodide stain by rinsing slides well with phosphate buffered saline at room temperature and stain for 10 seconds at room temperature. Wash slide three times with 100-250 ml phosphate buffered saline at room temperature and mount cover slip.

[0279] The immunofluorescence assay described above can be used to determine the presence of pANCA in DNase-treated, fixed neutrophils, e.g., by the presence of a pANCA reaction in control neutrophils (i.e., fixed neutrophils that have not been DNase-treated) that is abolished upon DNase treatment or by the presence of a pANCA reaction in control neutrophils that becomes cytoplasmic upon DNase treatment.

Example 3. Determination of ASCA Levels.

[0280] This example illustrates the preparation of yeast cell well mannan and an analysis of ASCA levels in a sample using an ELISA assay.

[0281] Yeast cell wall mannan may be prepared as described in Faille et ai, Eur. J. Clin. Microbiol. Infect. Dis., 1 1 :438-446 ( 1992) and in Kocourek et al, J. Bacteriol. , 100: 1 175- 1 181 (1969). Briefly, a lyophilized pellet of yeast Saccharomyces uvarum is obtained from the American Type Culture Collection (#38926). Yeast are reconstituted in 10 ml 2 x YT medium, prepared according to Sambrook et al., In "Molecular Cloning," Cold Spring Harbor Laboratory Press (1989). S. uvarum are grown for two to three days at 30°C. The terminal S. uvarum culture is inoculated on a 2 x YT agar plate and subsequently grown for two to three days at 30°C. A single colony is used to inoculate 500 ml 2 x YT media, and grown for two to three days at 30°C. Fermentation media (pH 4.5) is prepared by adding 20 g glucose, 2 g bacto-yeast extract, 0.25 g MgS0₄, and 2.0 ml 28% H₃P0₄ per liter of distilled water. The 500 ml culture is used to inoculate 50 liters of fermentation media, and the culture fermented for three to four days at 37°C.

[0282] S. uvarum mannan extract is prepared by adding 50 ml 0.02 M citrate buffer (5.88 g/1 sodium citrate; pH 7.0 ± 0.1 ) to each 100 g of cell paste. The cell/citrate mixture is autoclaved at 125°C for ninety minutes and allowed to cool. After centrifuging at 5000 rpm for 10 minutes, the supernatant is removed and retained. The cells are then washed with 75 ml 0.02 M citrate buffer and the cell/citrate mixture again autoclaved at 125°C for ninety minutes. The cell/citrate mixture is centrifuged at 5000 rpm for 10 minutes, and the supernatant is retained.

[0283] In order to precipitate copper/mannan complexes, an equal volume of Fehling's Solution is added to the combined supernatants while stirring. The complete Fehling's solution is prepared by mixing Fehling's Solution A with Fehling's Solution B in a 1 : 1 ratio just prior to use. The copper complexes are allowed to settle, and the liquid decanted gently from the precipitate. The copper/mannan precipitate complexes are then dissolved in 6-8 ml 3N HC1 per 100 grams yeast paste.

[0284] The resulting solution is poured with vigorous stirring into 100 ml of 8: 1 methanokacetic acid, and the precipitate allowed to settle for several hours. The supernatant is decanted and discarded, then the wash procedure is repeated until the supernatant is colorless, approximately two to three times. The precipitate is collected on a scintered glass funnel, washed with methanol, and air dried overnight. On some occasions, the precipitate may be collected by centrifugation at 5000 rpm for 10 minutes before washing with methanol and air drying overnight. The dried mannan powder is dissolved in distilled water to a concentration of approximately 2 g/ml.

[0285] A S. varum mannan ELISA may be used to detect ASCA. S. uvarum mannan ELISA plates are saturated with antigen as follows. Purified S. uvarum mannan prepared as described above is diluted to a concentration of 100 μg/ml with phosphate buffered saline/0.2% sodium azide. Using a multi-channel pipettor, 100 μΐ of 100 g/ml S. uvarum mannan is added per well of a Costar 96-well hi-binding plate (catalog no. 3590; Costar Corp., Cambridge, Mass.). The antigen is allowed to coat the plate at 4°C for a minimum of 12 hours. Each lot of plates is compared to a previous lot before use. Plates are stored at 2- 8°C for up to one month.

[0286] Patient sera may be analyzed in duplicate for ASCA-IgA or ASCA-IgG reactivity. Microti ter plates saturated with antigen as described above are incubated with phosphate buffered saline/0.05% Tween-20 for 45 minutes at room temperature to inhibit nonspecific antibody binding. Patient sera are subsequently added at a dilution of 1 : 80 for analysis of ASCA-IgA and 1 :800 for analysis of ASCA-IgG and incubated for 1 hour at room temperature. Wells are washed three times with PBS/0.05% Tween-20. Then, a 1 : 1000 dilution of alkaline phosphatase-conjugated goat anti-human IgA (Jackson Immunoresearch; West Grove, Pa.) or a 1 : 1000 dilution of alkaline phosphatase-conjugated goat anti-human IgG F(ab')₂ (Pierce; Rockford, 111.) is added, and the microtiter plates are incubated for 1 hour at room temperature. A solution of p-nitrophenol phosphate in diethanolamine substrate buffer is added, and color development is allowed to proceed for 10 minutes. Absorbance at 405 nm is analyzed using an automated EMAX plate reader (Molecular Devices; Sunnyvale, Calif.).

[0287] ASCA levels (e.g., IgG, IgA, or both) may be determined relative to a standard consisting of pooled sera obtained from patients with an established diagnosis of Crohn's disease (CD). Results with test patient samples are expressed as ELISA units and may be expressed as a percentage of the standard binding of the reference CD sera. Sera with circulating ASCA levels exceeding the reference range value may also be termed ASCA positive, whereas numerical values that are below the reference range may also be termed ASCA negative.

Example 4. Determination of Anti-OmpC Antibody Levels.

[0288] This example illustrates the preparation of OmpC protein and an analysis of anti- OmpC antibody levels in a sample using an ELISA assay.

[0289] The following protocol describes the purification of OmpC protein using spheroplast lysis. OmpF/OmpA-mutant E. coli are inoculated from a glycerol stock into 10- 20ml of Luri a Bertani broth supplemented with 100μg/ml streptomycin (LB-Strep; Teknova; Half Moon Bay, CA) and cultured vigorously at 37°C for about 8 hours to log phase, followed by expansion to 1 liter in LB-Strep over 15 hours at 25°C. The cells are harvested by centrifugation. If necessary, cells are washed twice with 100ml of ice cold 20mM Tris-Cl, pH 7.5. The cells are subsequently resuspended in ice cold spheroplast forming buffer (20mM Tris-Cl, pH 7.5; 20% sucrose; 0.1M EDTA, pH 8.0; 1 mg/ml lysozyme), after which the resuspended cells are incubated on ice for about 1 hour with occasional mixing by inversion. If required, the spheroplasts are centrifuged and resuspended in a smaller volume of spheroplast forming buffer (SFB). The spheroplast pellet is optionally frozen prior to resuspension in order to improve lysis efficiency. Hypotonic buffer is avoided in order to avoid bursting the spheroplasts and releasing chromosomal DNA, which significantly decreases the efficiency of lysis. [0290] The spheroplast preparation is diluted 14-fold into ice cold l OmM Tris-Cl, pH 7.5 containing l mg/ml DNasel and is vortexed vigorously. The preparation is sonicated on ice 4 x 30 seconds at 50% power at setting 4, with a pulse "On time" of 1 second, without foaming or overheating the sample. Cell debris is pelleted by centrifugation and the supernatant is removed and clarified by centrifugation a second time. The supernatant is removed without collecting any part of the pellet and placed into ultracentrifuge tubes. The tubes are filled to 1.5mm from the top with 20mM Tris-Cl, pH 7.5. The membrane preparation is pelleted by ultracentrifugation at 100,000 x g for 1 hr at 4°C in a Beckman SW 60 swing bucket rotor. The pellet is resuspended by homogenizing into 20mM Tris-Cl, pH 7.5 using a 1 ml pipette tip and squirting the pellet closely before pipetting up and down for approximately 10 minutes per tube. The material is extracted for 1 hr in 20mM Tris-Cl, pH 7.5 containing 1 % SDS, with rotation at 37°C. The preparation is transferred to ultracentrifugation tubes and the membrane is pelleted at 100,000 x g. The pellet is resuspended by homogenizing into 20mM Tris-Cl, pH 7.5 as before. The membrane preparation is optionally left at 4°C overnight.

[0291] OmpC is extracted for 1 hr with rotation at 37°C in 20mM Tris-Cl, pH 7.5 containing 3% SDS and 0.5 M NaCl. The material is transferred to ultracentrifugation tubes and the membrane is pelleted by centrifugation at 100,000 x g. The supernatant containing extracted OmpC is then dialyzed against more than 10,000 volumes to eliminate high salt content. SDS is removed by detergent exchange against 0.2% Triton. Triton is removed by further dialysis against 50mM Tris-Cl. Purified OmpC, which functions as a porin in its trimeric form, is analyzed by SDS-PAGE. Electrophoresis at room temperature results in a ladder of bands of about 100 kDa, 70 kDa, and 30 kDa. Heating for 10- 15 minutes at 65- 70°C partially dissociates the complex and results in only dimers and monomers (i.e., bands of about 70 kDa and 30 kDa). Boiling for 5 minutes results in monomers of 38 kDa.

[0292] The OmpC direct ELISA assays may be performed essentially as follows. Plates (USA Scientific; Ocala, FL) are coated overnight at 4°C with Ι ΟΟμΙ/well OmpC at 0.25μg/ml in borate buffered saline, pH 8.5. After three washes in 0.05% Tween 20 in phosphate buffered saline (PBS), the plates are blocked with 150μ1Λνε11 of 0.5% bovine serum albumin in PBS, pH 7.4 (BSA-PBS) for 30 minutes at room temperature. The blocking solution is then replaced with Ι ΟΟμΙ/well of Crohn's disease or normal control serum, diluted 1 : 100. The plates are then incubated for 2 hours at room temperature and washed as before.

Alkaline phosphatase-conjugated goat anti-human IgA (a-chain specific), or IgG (γ-chain specific) (Jackson ImmunoResearch; West Grove, Pa.) is added to the plates at a dilution of 1 : 1000 in BSA-PBS . The plates are incubated for 2 hours at room temperature before washing three times with 0.05% Tween 20/PBS followed by another three washes with Tris buffered normal saline, pH 7.5. Substrate solution (1.5mg/ml disodium p-nitrophenol phosphate (Aresco; Solon, Ohio) in 2.5mM MgCl₂, 0.01 M Tris, pH 8.6) is added at

Ι ΟΟμΙ/well, and color is allowed to develop for one hour. The plates are then analyzed at 405 nm.

[0293] Anti-OmpC antibody levels may be determined relative to a standard consisting of pooled sera obtained from patients with an established diagnosis of Crohn' s disease (CD). Sera with circulating anti-OmpC antibody levels exceeding the reference range value may also be termed anti-OmpC antibody positive, whereas numerical values that are below the reference range may also be termed anti-OmpC antibody negative. In certain instances, anti- OmpC antibody positive reactivity may be defined as reactivity greater than two standard deviations above the mean reactivity obtained with control (normal) sera analyzed at the same time as the test samples.

Example 5. Determination of Anti-I2 Antibody Levels.

[0294] This example illustrates the preparation of recombinant 12 protein and an analysis of anti-I2 antibody levels in a sample using an ELISA assay or a histological assay.

[0295] The full-length 12-encoding nucleic acid sequence may be cloned into the GST expression vector pGEX. After expression in E. coli, the protein is purified on a GST column. The purified protein may be shown to be of the expected molecular weight by silver staining, and may be shown to have anti-GST reactivity upon Western blot analysis. The full-length 12-encoding nucleic acid sequence may also be cloned into a Hex-His6 expression vector, expressed in E. coli, and the resulting protein purified.

[0296] Human IgA and IgG antibodies that bind the GST-I2 fusion polypeptide may be detected by direct ELISA assays essentially as follows. Plates (Immulon 3; DYNEX

Technologies; Chantilly, VA) are coated overnight at 4°C with 100 μΐ/well GST-I2 fusion polypeptide (5 g/ml in borate buffered saline, pH 8.5). After three washes in 0.05% Tween 20 in phosphate buffered saline (PBS), the plates are blocked with 150 μΐ/well of 0.5% bovine serum albumin in PBS, pH 7.4 (BSA-PBS) for 30 minutes at room temperature. The blocking solution is then replaced with 100 μΐ/well of CD serum, ulcerative colitis (UC) serum, or normal control serum, diluted 1 : 100. The plates are then incubated for 2 hours at room temperature and washed as before. Alkaline phosphatase-conjugated secondary antibody (goat anti-human IgA (a-chain specific); Jackson ImmunoResearch; West Grove, Pa.) is added to the IgA plates at a dilution of 1 : 1000 in BSA-PBS. For IgG reactivity, alkaline phosphatase conjugated secondary antibody (goat anti-human IgG (γ-chain specific); Jackson ImmunoResearch) is added. The plates are incubated for 2 hours at room

temperature before washing three times with 0.05% Tween 20/PBS followed by another three washes with Tris buffered normal saline, pH 7.5. Substrate solution ( 1 .5 mg/ml disodium p- nitrophenol phosphate (Aresco; Solon, Ohio) in 2.5 mM MgCl₂, 0.01 M Tris, pH 8.6, is added at 100 μΐ/well, and color allowed to develop for one hour. The plates are then analyzed at 405 nm. Nonspecific binding of sera to the control GST protein (typically <0.1 ) are subtracted from raw values of 12 binding to obtain I2-specific absorbances.

[0297] Anti-I2 antibody levels may be determined relative to a standard consisting of pooled sera obtained from patients with an established diagnosis of Crohn's disease (CD). Sera with circulating anti-I2 antibody levels exceeding the reference range value may also be termed anti-I2 antibody positive, whereas numerical values that are below the reference range may also be termed anti-I2 antibody negative. In certain instances, anti-I2 antibody positive reactivity may be defined as reactivity greater than two standard deviations above the mean reactivity obtained with control (normal) sera analyzed at the same time as the test samples.

[0298] For histological analysis, rabbit anti-I2 antibodies may be prepared using purified GST-I2 fusion protein as the immunogen. GST-binding antibodies are removed by adherence to GST bound to an agarose support (Pierce; Rockford, IL), and the rabbit sera validated for anti-I2 immunoreactivity by ELISA analysis. Slides are prepared from paraffin- embedded biopsy specimens from CD, UC, and normal controls. Hematoxylin and eosin staining are performed, followed by incubation with I2-specific antiserum. Binding of antibodies is detected with peroxidase-labeled anti-rabbit secondary antibodies (Pierce; Rockford, 111.). The assay may be optimized to maximize the signal to background and the distinction between CD and control populations.

Example 6. Genotyping for Three Crohn's Disease Associated Variants of NOD2.

[0299] This example shows a genotyping assay that can be used to detect the presence or absence of a NOD2 variant. [0300] Genotyping may be performed using a genotyping assay employing 5'-exonuclease technology, the TaqMan MGB™ assay (PE Biosystems; Foster City, CA). Primers may be designed using the software PrimerExpress 1.5.™ (PE Biosystems) and sequence information may be found in dbSNP for NOD2 variants R702W ("SNP 8"), G908R ("SNP 12"), and 1007fs ("SNP 13"). The MGB™ design adds a "minor groove binder" to the 3' end of the TaqMan™ probes, thereby increasing the binding temperature of the probe and enabling the use of shorter probes than in conventional TaqMan™ assays (Kutyavin et al., Nucleic Acids Res. , 25:3718-3723 (1997)). This has the effect of increasing the discrimination between the alleles in the assay (Kutyavin et al, Nucleic Acids Res., 28:655-661 (2000)). Assays may be performed following the manufacturer's recommendations (PE Biosystems bulletin 4317594) in an ABI 7900 instrument. Genotyping is typically performed blinded to clinical status of the subjects. Exemplary primers and probes suitable for use in the NOD2 genotyping assay are shown in Tables 3 and 4.

Table 3

Table 4

Example 7. Methods for Predicting Post-Surgery Risk Associated with Ileal Pouch- Anal Anastomosis (IPAA).

[0301] This example illustrates the use of multiple markers for providing prognostic information to physicians in assessing patients faced with ileal pouch-anal anastomosis (IPAA) and dysplasia at the time of colectomy.

[0302] In particular embodiments, this example demonstrates that an individual's risk of developing dysplasia/cancer and/or inflammatory complications following a surgical procedure such as ΓΡΑΑ can be determined using an algorithmic approach based upon detecting the presence, level, or genotype of a combination of markers in a serum sample from the individual. For instance, this example shows that the detection of multiple markers, whether they are serological (e.g., ASCA IgA, ASCA IgG, anti-OmpC, anti-CBirl , anti-I2, and/or pANCA), protein {e.g., TNF alpha, IL-6, IL-8, IL-12, IL- 17, IL-23, C reactive protein (CRP), EGF, and/or serum amyloid A (SAA)), or genetic {e.g., NOD2 SNPs R702W,G908R, and/or 1007fs), can aid physicians in determining those individuals at risk of developing dysplasia and/or complications post-surgery following an IPAA procedure.

[0303] FIG. 2 illustrates an exemplary prognostic marker profile using a combination of the above markers for determining an individual's post-surgery risk following a surgical procedure whereby the colon is removed and an internal pouch is created {e.g., IPAA).

[0304] FIG. 3 illustrates (1 ) the concentration levels of ANCA, ASCA IgA, ASCA IgG, anti-CBirl , anti-OmpC, C reactive protein (CRP), serum amyloid A (SAA), EGF, and anti-I2, and (2) the presence or absence of pANCA, that were detected using preoperative patient serum samples for assessing the risk of these patients in developing complications such as pouchitis and/or dysplasia following a surgical procedure whereby the colon is removed and an internal pouch is created {e.g., IPAA).

Example 8. Methods for Prognosing various Risks Associated with Ileal Pouch-Anal Anastomosis (IPAA).

[0305] This example illustrates the use of one or more of the markers described herein for providing prognostic information to physicians in assessing patients faced with ileal pouch- anal anastomosis (IPAA) and dysplasia.

Methodology

[0306] Sample population: N = 195 (duplicates removed). Final diagnosis and number of samples for each diagnosis follows:

Table 5

Final Dx: 0 = nl; 1 = IPS; 2 = Active pouchitis; 3 = Refractory pouchitis; 4 = CD of the pouch; 5 = Cuffitis; 6 = Surgical complication; and 7 = Anismus; IPS = Irritable Pouch Syndrome; nl = Normal Pouch.

[0307] Seven (7) of the markers analyzed in FIG. 3 {e.g., ANCA, ASCA IgA, ASCA IgG, anti-CBirl antibody, anti-OmpC antibody, anti-I2 antibody, and pANCA) were reviewed for further analysis and compared to the final diagnosis to determine patterns.

[0308] The outputs examined were as follows:

1 . Cancer/Dysplasia vs Non-cancer;

2. CD vs Other pouch outcomes; (4 vs 0-3 and 5-7 final. dx)

3. CD vs Surgical complications; (4 vs 6 final.dx)

4. CD phenotype. (4 final.dx)

[0309] For each of the 4 outputs above, the 7 markers were analyzed and grouped two different ways: (1 ) above/below median concentration levels; and (2) in four concentration quadrants. pANCA is either present or not present; thus, no quadrant analysis was performed with this marker. For median analysis, each marker was classified as either below median, e.g., lower than a reference value {e.g., a median concentration level), or above median, e.g., higher than a reference value {e.g., a median concentration level). For quadrant analysis, each marker was scored into 1 of 4 quartiles (Q1 -Q4). The quartile scores for a combination of markers may be summed to produce a quartile sum score (QSS). p values were calculated with a Fisher's exact test method and are listed for each marker. The results are tabulated below.

Cancer vs Non-cancer: Table 6A

Cancer vs Non-cancer: Table 6B

All other vs CD: Table 7A

All other vs CD: Table 7B

CD vs Surg Complications Table 8A

CD vs Surg Complications Table 8B

16] CD Phenotvpes Table 9 A

[0317] CD Phenotypes Table 9B

Inflamm. = inflammatory; fibrost. = fibrostenosing; and fist. = fistulating.

[0318] In view of the above analyses, the following conclusions can be reached.

• The data in Tables 6 A&B indicates OmpC is a good marker for prognosticating between cancer and non-cancer.

• The data in Tables 7 A&B indicates that ANCA, ASCA-IgG, CBir-1 , OmpC, pANCA and 12 can be used to prognosticate between CD versus other indications. • The data in Tables 8 A&B indicates ANCA is a good marker to prognosticate between CD and surgical complications.

• The data in Tables 9 A&B indicates CBir- 1 and OmpC are good markers to be used to prognosticate CD phenotype.

[0319] In certain embodiments of this invention, the quartile measurements of particular markers within a given population are summed. Quartiles are a set of "cut points" that divide a sample of data into groups containing (as far as possible) equal numbers of observations. The lower quartile is the data value a quarter way up through the ordered data set; the upper quartile is the data value a quarter way down through the ordered data set. In quartile analysis, there are three numbers (values) that divide a range of data into four equal parts. The first quartile (also called 'lower quartile') is the number below which lies the 25 percent of the bottom data. The second quartile (the 'median') divides the range in the middle and has 50 percent of the data below it. The third quartile (also called 'upper quartile') has 75 percent of the data below it and the top 25 percent of the data above it.

[0320] The analysis above involves detecting or determining the presence and/or level (e.g., magnitude) of one or more prognostic markers of interest using quartile analysis. In this type of statistical analysis, the level of a marker of interest is defined as being in the first quartile (0-25%), second quartile (25-50%), third quartile (50-75%), or fourth quartile (75- 100%) in relation to a reference database of samples. A non-limiting example of quartile analysis suitable for use in the present invention is described in, e.g., Mow et ah,

Gastroenterology, 126:414-24 (2004).

Example 9. Protocol for the Purification and Refolding of GST-I2.

1.0 PURPOSE

[0321] This example describes a procedure for the purification and refolding of the GST-I2 antigen from frozen bacterial glycerol stock. This process will take eight days to complete.

2.0 SCOPE

[0322] The rGST-I2 Antigen Prep is the antigen used to capture antibodies to Psuedomonas fluorescens-rdated peptide in the serum of patients with Crohn's Disease (CD) as described in Example 20. 3.0 PRINCIPLE

[0323] The purpose of the rGST-I2 Antigen Prep procedure is to purify and refold the GST-I2 so it can be further purified from bacterial contaminants. The refolding process allows the antigen to be purified and allows it to properly interact with anti-GST antibodies in the 12 ELISA.

4.0 DEFINITIONS

[0324] GST-I2: Glutathione S-transferase fused to Pseudomonas fluorescens-rdated peptide.

5.0 PROCEDURE

5.1. DAY 1

5.1.1. Prepare the overnight culture. Start with 60mls of LB media that has been autoclave sterilized in a 250ml Erlenmeyer flask. Label this flask with "12 Antigen overnight culture" and today's date.

5.1.2. Pre-warm this media in the incubator shaker that has been set at 37°C.

Warm the media for 30 minutes.

5.1.3. While the media is incubating pull one aliquot of Ampicillin

(50mg/ml) out of the -70°C freezer and thaw it out at room

temperature.

5.1.4. Add 60μ1 of 50mg/ml Ampicillin to the pre-warmed media. Let the media mix in the incubator for 5 minutes at 200rpm.

5.1.5. Remove the GST-I2 Glycerol Stock from the -70°C freezer and place it directly in a bucket of dry ice. Do not allow the glycerol stock to thaw.

5.1.6. Inoculate the LB/Ampicillin media with the frozen GST-I2 glycerol stock. Turn the incubator/shaker off. While still keeping the glycerol stock on dry ice, open the cap of the glycerol stock tube. Using an inoculation loop scrape the surface of the frozen GST-I2 glycerol stock. Slightly remove the aluminum foil from the top of the 250ml flask that contains the LB/Ampicillin media. Just open the foil top enough to the point where you can fit an inoculation loop inside. Place the inoculation loop (with glycerol scrapings) in the flask and slowly move it around for a few seconds. Remove the inoculation loop and secure the foil on the flask.

5.1.7. Turn the shaker on and let the culture incubate over night at 200rpm and 37°C.

5.1.8. Take this time to set up the LB media that will be used the following day. Take two 500ml volumes of LB media that has been autoclaved sterilized in 2L Erlenmeyer flasks and place them in a 37°C incubator overnight with no shaking. Be sure to place them in an incubator that is not being used for the overnight culturing of the glycerol stock. This is being done to ensure that pre-warmed media will be ready for use the next day.

2

Check the two 500ml volumes of LB media that have been warming overnight. They should still be clear with no visible growth.

Next, check the OD 600 of the overnight culture using the Nanospec spectrophotometer. Use 1 ml of LB media as your blank. Use 1 ml of overnight culture to check the OD 600. The OD 600 of the overnight culture should be around 1.9-2.3. If it is not in this range discard the culture and start again.

Place the two 500ml volumes of pre-warmed LB media in the same incubator shaker that contains the overnight culture. Next, pull out two aliquots of Ampicillin (50mg/ml) from the -70°C freezer and thaw them out to room temperature.

Add 500μ1 of Ampicillin to each of the 500ml volumes of LB Media. Let these flasks shake in the incubator for 5mins at 37°C and 200rpm. Next, perform a 1 :20 dilution of the overnight culture into both of the 500ml volumes of LB/ Amp media. This is done by adding 25ml of the overnight culture to 500ml of the LB/Amp media. At this point each 500ml culture will be designated as either culture A or B. Label both cultures with today's date. In addition, label each culture as either "GST-I2 culture A" or "GST-I2 culture B".

Incubate these cultures for lhr at 37°C and 200rpm.

After 1 hour of incubating, check the OD 600 of both of the cultures. The OD 600 of the cultures needs to reach 0.6-0.9 before protein expression can be induced with isopropyl β-D-l -thiogalactopyranoside (IPTG). If your initial 1 hour OD 600 reading is under 0.6, continue to check the OD 600 of the culture every 15mins until the OD 600 reaches the range of 0.6-0.9. Record the OD 600 of each culture at this point.

Once the OD 600 of the cultures have reached the accepted range for IPTG induction a 1 ml aliquot of each culture is taken and placed into a 1.5ml micro centrifuge tube. Each tube is labeled with today's date, "GST-I2 culture A/B", and T=0. Place these two T=0 aliquots on ice. It is important to take these aliquots before IPTG induction because they will be used for later gel analysis. Induce expression of the GST-I2 antigen using I mM IPTG. Add 500μ1 of 1 M IPTG solution to each 500ml culture. Note the time that the cultures were induced.

Incubate these cultures for 4 hours at 37°C and 200rpm. The following procedures 5.2.1 1 . - 5.2.14. should be performed during this incubation process.

Take the 1ml T=0 culture aliquots that have been sitting on ice and place them into a centrifuge (Eppendorf 5402 centrifuge). Spin the aliquots at 5,000xg for lOmins at 4°C. Remove the supernatant carefully without disturbing the bacterial pellet. Store the pellets at - 70°C.

Label two 500ml centrifuge bottles with today's date and "GST-I2 Bacterial Pellet A/B". Weigh each bottle and record their mass in grams.

Place the weighed bottles on ice right before the 4 hour incubation period is over.

Label two micro centrifuge tubes with today's date, "GST-I2 culture A/B", and T=4hr. Place these two T=4hr tubes on ice.

When the 4 hour time point is reached, turn off the incubator. Record the OD 600 of each culture.

Take a 1ml aliquot of each culture and place it into the appropriately labeled micro centrifuge tube. Place the aliquots on ice.

Pour the remaining volume of each culture into the appropriately labeled 500ml centrifuge bottle. Place these bottles into the Sorvall RC-3B centrifuge with H-6000 rotor.

Centrifuge the cultures for 10 minutes at 5,000xg and 4°C.

Remove the bottles from the centrifuge and place them on ice. Empty the supernatant into the 2L Erlenmeyer flasks that were used for culturing. These will be used as waste containers. Remove all the supernatant while keeping the bacterial pellet intact at the bottom of the bottle. Place the bottles back on ice.

Weigh the bottles to determine the mass of the bottles plus the bacterial pellets. Use a kim wipe to wipe off the excess moisture from the outer surface of the bottles. This will allow for a more accurate mass determination.

Determine the weight of the bacterial pellet by subtracting the values determined in step 5.2.12. from the values in step 5.2.20.

Write the weight of the pellet on each of the bottles and store them at - 70°C.

I l l 5.2.23. Take the 1 ml aliquots from step 5.2. 16. and place them into a centrifuge (Eppendorf 5402 centrifuge). Spin the aliquots at 5,000xg for l Omins at 4°C. Remove the supernatant carefully without disturbing the bacterial pellet. Store the pellets at -70°C.

DAY 3

5.3.1. Steps 5.3.9. through 5.3.5 1. will require the making of fresh reagents and take approximately 6 hours to complete.

5.3.2. An SDS-PAGE protein gel must be run to confirm that the protein expression of the GST-I2 antigen was induced. This must be done before any further steps can be carried out.

5.3.2.1. Remove the 1 ml bacterial pellet time points (T=0,4hr) from the -70°C freezer and place them on ice.

5.3.2.2. Suspend the pellets in Nanopure distilled H₂0, as follows

The amount of H₂0 that will be used for suspension is based on the OD 600 of each time point.

5.3.2.3. To determine the amount of H₂0 to add to the pellet of each time point, plug the OD 600 of each time point into the following equation.

Water to add to pellet = ((OD600)/0.418)* 192)/2

Record the volume of H₂0 added to suspend each bacterial pellet time point.

5.3.3. Label 4 new 1 .5ml micro centrifuge tubes with the time points shown above in 10.3.2.3. These new tubes will be used to prepare the samples for gel analysis.

5.3.4. Into each new micro centrifuge tube load 29μ1 of its respective

bacterial pellet suspension. Next, load l Oul of 4x sample buffer into each micro centrifuge tube. Then, load 1 μΐ of 2.5% beta- mercaptoethanol. Mix each tube and then incubate them in a heat block at 90°C for 5minutes.

5.3.5. Prepare the 4-12% Bis-Tris gel. Refer to the instructions of the XCell SureLock™ Mini Cell (Invitrogen - Part number EI0001 ) for setting up the gel.

http://tools.invitrogen.com/content/sfs/manuals/surelock man.pdf

5.3.5.1. Manual Pgs 9- 13, 17. The running buffer that is used in this process is MES SDS Running Buffer.

5.3.6. Load 30μ1 of each sample into separate wells. Load ΙΟμΙ of the

Protein Ladder in a separate well. Run the gel for 35 minutes at 200 volts. Remove the gel from its casing and place it in a flat bottom container. Empty pipette tip box lids work well . Briefly wash the gel for 15 seconds with 50mls of Nanopure distilled H₂0. Then add l OOmls of Simply Blue Safe Stain and incubate the gel on a rocker for 50mins. Decant the Simply Blue Safe Stain and add 100ml of Nanopure distilled H₂0. Place the gel back on a rocker and incubate it for 1 hour. Within lOmins of this incubation step you'll be able to confirm that the GST-I2 antigen was expressed (Figure 4A). When the bands are confirmed move on to the next step.

Take this time to prepare 50mls of 12 Buffer A - 50mM Tris-CL, 0.5mM EDTA, 5% glycerol, 5mM DTT, pH 8.0.

Take this time to prepare 50mls of 12 Denaturing Buffer - l OmM Tris- Cl, 0.1M NaH₂P0₄, 8M Urea, 5mM DTT, pH 8.0 This solution is stored at room temperature until use.

Take this time to prepare 400mls of 12 Refolding Buffer - 25mM Tris- Cl, lOOmM NaCl, 10% glycerol, 0.2M Urea, 0.5mM oxidized glutathione (GSSG), I mM reduced glutathione (GSH), pH 9.0. This solution is chilled in a 1L beaker on wet ice until use. Place parafilm over the beaker to prevent contamination.

Prepare 20mls of bacterial lysis buffer in a plastic 50ml conical tube. Label the tube as "12 Bacterial Lysis Buffer" (as follows in steps 5.3.13 through 5.3.16.).

Add 20mls of 12 Buffer A to the 50ml conical tube.

Add 20mg of Lysozyme to the 50ml conical tube (final concentration 1 mg/ml).

Add one Complete Protease Inhibitor Tablet (Roche) to the 50ml conical tube.

Vortex mix the contents of the 50ml conical tube until the Lysozyme and Protease Inhibitor Tablet are in solution. Then place this tube on wet ice until needed. It must be chilled before use.

Remove the 12 bacterial pellet(s) from the -70°C freezer and place it on ice. The wet weight of the 12 bacterial pellet(s) must be between 3-5g. Multiple pellets may need to be used to achieve the 3-5g mass range. Thaw the pellet(s) out on ice for 15 minutes.

While the bacterial pellet is thawing prepare the 20ml of 2%

Deoxycholate (DOC) Reagent. Label a plastic 50ml conical tube with "12 2% DOC Reagent". Add the following to the 50ml conical tube. Add 20ml of 12 Buffer A.

Add 400mg of Sodium Deoxycholate (DOC). Vortex the solution briefly then mix the components end over end until the DOC is completely dissolved. This solution is kept at room temperature until use.

Add 20mls of 12 Bacterial Lysis Buffer to the bacterial pellet on ice. Suspend the pellet thoroughly on ice with a 10ml serological pipet. The suspension is performed in the 500ml centrifuge bottle and finished once there is no sign of visible particulates.

Incubate the suspension on ice for 30 mins.

Transfer the suspension to a 50 ml conical tube. Label this tube "12 Total Lysate Sonicate" and today's date. Put this tube on ice.

Sonicate the suspension preparation on ice.

Adjust the Amplitude to 40%.

Push the recall button and select Program ID #1. Then press Enter. This program is set to perform 1 second pulses in one 10 sec cycle. Insert the small-tipped probe in the 50ml conical tube. Make sure the probe is inserted well into the lysate suspension. Make sure the tip is not touching the surface of the 50ml conical tube.

Press start to send the suspension through one cycle of sonication. Then let the suspension sit for 15 seconds to prevent overheating of the sample.

Repeat step 5.3.29. five times. Then check the suspension with a 10 ml serological pipet. Run the suspension through the pipet several times. The suspension should run like a fluid out of the tip of the pipet with no visible changes in viscosity or stickiness. If the sample appears gooey that means the genomic DNA has not been sufficiently broken down. If this occurs, repeat step 5.3.29. until this trait disappears.

Transfer ~20ml of the sonicated sample into a 50ml Centrifuge Tube (Nalgene). After the transfer, save 50μ1 of the sonicate for later gel analysis. This should be kept in the original 50ml centrifuge tube labeled with "12 Total Lysate Sonicate" until it is needed. Store the 50μ1 aliquot at 4°C.

Place the sonicate suspension in the Beckmann J2-21 centrifuge and spin the sample at 12,000xg ( 12,400rpm in JA-20 rotor) at 4°C for 10 minutes.

After centrifugation, decant the supernatant into a 50ml conical tube labeled with "S I Sonicate Supernatant" and today's date. This supernatant will be used for later gel analysis. Store it at 4°C until it is needed. At this point the insoluble pellet will undergo further processing. Suspend the insoluble pellet in 20mls of 12 2% DOC Reagent in the 50ml centrifuge tube. Suspend the pellet thoroughly at room temperature with a 10ml serological pipet. The suspension is not finished until there is no visible sign of particulates. This step will take approximately 10 minutes to perform.

Incubate the 2% DOC suspension for 30 minutes at room temperature. Place the 2% DOC suspension in the Beckmann J2-21 centrifuge and spin the sample at 12,000xg ( 12,400rpm in JA-20 rotor) at 4°C for 10 minutes.

After centrifugation, decant the supernatant into a 50ml centrifuge tube labeled with "S2 DOC Wash Supernatant" and today's date. This supernatant will be used for later gel analysis. Store it at 4°C until it is needed. At this point the insoluble pellet will undergo further processing.

Gently wash the insoluble pellet with 20mls of I xPBS pH 7.4. This will be performed twice with 10ml increments of I PBS pH 7.4 Slowly add lOmls of I xPBS pH 7.4 to the 50ml centrifuge bottle. Do not disturb the pellet. Cap the bottle and turn the bottle onto its side. Slowly rotate the bottle several times.

Decant the I PBS wash.

Repeat step 5.3.39.

Decant the I PBS wash.

Use a 1ml pipette (P1000) to remove any residual liquid.

The pellet is then solubilized with 20mls of 12 Denaturing Buffer in the 50ml centrifuge tube. Suspend the pellet thoroughly at room temperature with a 10ml serological pipet. This solubilization step will take approximately 15 minutes. To sufficiently solubilize this pellet, make sure the tip of the pipette is pressed firmly against the centrifuge tube wall while mixing. This will create a greater shearing force to further break down and solubilize the pellet. You will not completely solubilize the pellet but the particulates should be broken down to the point where their diameters are no larger than 1 millimeter.

This mixture is then incubated at room temperature for 30 minutes. Place the solubilized mixture in the Beckmann J2-21 centrifuge and spin the sample at 12,000xg ( 12,400rpm in JA-20 rotor) at 4°C for 15 minutes.

Decant the supernatant into a 50ml conical tube labeled with

"Denatured GST-I2" and today's date. Save 50μ1 of this solution in a 1.5ml micro centrifuge tube for later gel analysis (Store at 4°C). Discard the pellet. The ~20ml of Denatured GST-I2 can be kept at room temperature until its need for the following step.

Dilute the Denatured GST-I2 20-fold slowly in ice cold 12 Refolding Buffer. The following steps are performed at 4°C in a refrigerator. A peristaltic pump is used to slowly add the supernatant to the ice cold refolding buffer that is being stirred on a magnetic stir plate. The flow rate of the addition of the supernatant is approximately 0.5ml/min. The flow rate may shift as long as the drop of supernatant is fully dissolved in the refolding solution before the addition of the subsequent drop. This is done to optimize the dilution of the GST-I2 and to prevent any steric hindrance that could occur during refolding due the GST-I2 molecules being too close together.

Priming the pump (priming can be performed during the 30 min incubation steps in 5.3.35. and 5.3.45. to conserve time).

5.3.49.1. Place the peristaltic pump with attached tygon tubing in the refrigerator. Next to the pump place a magnetic stir plate. Use a stir plate with an electronic read-out so you can accurately determine the rpm during the dilution step.

5.3.49.2. Prime the pump with 40mls of Nanopure Distilled H₂0.

Place the tubing that is attached to the "in" connector into a 50ml conical tube containing 40 ml of H₂0. Make sure the tubing is at the bottom of the conical tube. Place the tubing that is attached to the "out" connector in a 250ml beaker. This beaker is a waste basin for the priming process. Set the dial on the peristaltic pump to 10 and put the pump on its prime setting. Click the "forward" button to begin priming.

5.3.49.3. Once the water priming is finished, prime the tubing with

20mls of Denaturing Buffer. Repeat the directions shown in step 5.3.49.2, except use 20mls of 12 Denaturing Buffer instead of water. After this step the pump is ready for the loading of the denatured GST-I2.

Loading Denatured GST-I2 onto the pump and subsequent dilution in 12 Refolding Buffer.

5.3.50.1. Make sure the pump is turned off.

5.3.50.2. Place the 50ml conical tube containing the Denatured GST- 12 in the refrigerator next to the peristaltic pump. Insert the tubing that is attached to the "in" connector of the peristaltic pump. Make sure the tubing reaches the bottom of the 50ml conical tube.

5.3.50.3. Place a magnetic stir bar in the 1L beaker that contains the 12

Refolding Buffer. Remove the beaker from the wet ice and place it on the magnetic stir plate in the refrigerator. Turn on the stir plate and adjust the rpm setting to 120.

5.3.50.4. Insert the tubing that is attached to the "out" connector of the pump into the 1 L beaker. Do not insert the tube into the refolding buffer. You want to have a gap (6-7cms) between the end of the tube and the surface of the refolding buffer. This will allow proper drop formation when the sample is being loaded into the refolded buffer. Make sure there is parafilm sealing the beaker.

5.3.50.5. Put the pump on its slow setting and then adjust the dial to 0.

Click the "forward" button to begin loading of the Denatured GST-I2.

5.3.50.6. The start time of the dilution process begins when the

sample reaches the end of the tube and begins dropping into the refolding buffer. At this point check to see that the drops are dissolving into solution before the next drop is added. If the drops are not dissolving quickly enough turn the stir bar speed up to 140rpm.

5.3.50.7. Once all the Denatured GST-I2 has been loaded into the beaker turn off the peristaltic pump. Label the beaker with "Refolded GST-I2" and today's date. Reduce the speed of the stir plate to l OOrpm and let the dilution mixture incubate overnight at 4°C.

Prepare 10 Liters of l xPBS pH 7.4 (2 x 5 Liter volumes).

5.3.51.1. Acquire two 5 Liter Beakers. Rinse them with 200ml of

Nanopure distilled H₂0 before use. Label each beaker with "l xPBS pH 7.4" and today' s date.

5.3.51 .2. Add 250ml of 20xPBS pH 7.4 to each 5 Liter Beaker.

5.3.51.3. Add 4.75L of Nanopure Distilled H20 to each 5 Liter

Beaker.

5.3.51.4. Mix each solution and cover each Beaker with aluminum foil. Store each solution in a 4°C refrigerator overnight.

Run a protein gel to determine if the GST-I2 has been processed properly up to the point where the GST-I2 is denatured in the

Denaturing Buffer.

The following samples are run on this Gel :

5.4.2.1. 12 Total Lysate Sonicate, Step 5.3.31. - TLS 5.4.2.2. S I Sonicate Supernatant, Step 5.3.33. - S I

5.4.2.3. S2 DOC Wash Supernatant, Step 5.3.37. - S2

5.4.2.4. Denatured GST-I2, Step 5.3.47. - DEN

Sample preparation: Label four micro centrifuge tubes with TLS, S I , S2 and DEN. Add the following to each tube:

5.4.3.1. Ι μΐ of Sample

5.4.3.2. 10μ1 οί 4Χ Sample Buffer

5.4.3.3. 1 μΐ of 2.5% beta-mercaptoethanol

5.4.3.4. 28μ1 of Nanopure distilled H₂0

Mix the samples and then incubate them in a heat block at 90°C for 5 minutes. Repeat step 5.3.5. through 5.3.7. to prepare and process the gel.

Decant the Simply Blue Safe Stain and add 100ml of Nanopure distilled H₂0. Place the gel back on a rocker and incubate it for 1 hour. Within 10 mins of this incubation step you'll be able to confirm that the GST-I2 antigen is present in the denatured sample (DEN) (Figure 4B). When the bands are confirmed move on to the next step.

Take three pieces of dialysis tubing (6-8mwco) that have been pre-cut to 36cm in length and submerge them in 500ml of Nanopure distilled H₂0. Let the dialysis tubing soak for 30 minutes.

Remove the "Refolded GST-I2" beaker from the refrigerator and place it on ice. The solution in the beaker will be clear with no visible precipitation. Remove a 50μ1 sample of this solution for gel analysis. Label this sample "Diluted rGST-I2" and today's date.

Remove one beaker containing 5 Liters of cold l xPBS pH 7.4 from refrigerator. Make sure this solution is mixed before placing it on ice. Label this beaker with "1 ^st Exchange".

Take three 50ml conical tubes and place them on ice. These conical tubes will be used to transfer the refolded GST-I2 into the dialysis tubing. Using a 25ml serological pipet, transfer approximately 47mls of refolded GST-I2 into each 50ml conical tube.

Remove one of the pieces of dialysis tubing from the distilled water and place a clamp on the bottom of the tubing. Make sure the clamp is fastened and that it covers the width of the tubing to ensure a tight seal. Insert a glass funnel into the open end of the dialysis tubing. Make sure that the funnel is inserted and that the inserted portion is held firmly against the dialysis tubing. It is important that this is done to prevent slippage of the tubing while it is being loaded. Due to the slippery nature of the sample that is being loaded, the tubing should be held firmly from the top at all times.

While the funnel is being held in position carefully load the refolded GST-I2 into the funnel. When each conical tube is emptied place it back on ice.

After the last conical tube of refolded GST-I2 is loaded place that empty conical tube on ice. With both hands, firmly grip the top of the dialysis tubing. Force all air bubbles out of the tubing, use a kim wipe to remove the bubbles. Make sure that there is no air left in the tubing before the top clamp is fastened.

Fasten the clamp on the top of the tubing. This tubing will expand after dialysis is finished so make sure that enough space is left in the tube for expansion. Leave 6cm of space from the clamp to the surface of the sample. This will allow for that expansion. Once again there should be no air in the tube. Make sure that there are no leaks present. Slowly place the full dialysis tube in the 5 Liter beaker of cold I xPBS pH 7.4.

Repeat steps 5.4.9. through 5.4.15. two more times. Use the same three 50ml conical tubes that were previously used. There will be a total of three filled dialysis tubes in the 5 Liter beaker at the end of this step.

Place the beaker on a magnetic stir plate in a 4°C refrigerator. Place a magnetic stir bar in the beaker. Set the stir plate to mix at l OOrpm. Make sure the stir bar is not hitting any of the dialysis tubes. Incubate these dialysis tubes for 4hrs at 4°C.

Slowly remove the dialysis tubes from the 5 liter beaker and place them in the other 5 liter beaker of I PBS pH 7.4 that has been stored in the 4°C. Make sure not to mix or agitate the solution inside of the dialysis tube. Place a magnetic stir bar in the beaker. Set the stir plate to mix at lOOrpm. Label this Beaker with "2^nd Exchange". Incubate these dialysis tubes for overnight at 4°C.

Prepare 10 Liters of I xPBS pH 7.4 (2 x 5 Liter volumes).

Acquire two 5 Liter Beakers. Rinse them with 200ml of Nanopure distilled H₂0 before use. Label each beaker with "I xPBS pH 7.4" and today's date.

Add 250ml of 20xPBS pH 7.4 to each 5 Liter Beaker.

Add 4.75L of Nanopure Distilled H20 to each 5 Liter Beaker.

Mix each solution and cover each Beaker with aluminum foil. Store each solution in a 4°C refrigerator overnight. Slowly remove the dialysis tubes from the 5 liter beaker and place them in the other 5 liter beaker of I xPBS pH 7.4 that has been stored in the 4°C. Make sure not to mix or agitate the solution inside of the dialysis tube. There may be some precipitation at this point. Place a magnetic stir bar in the beaker. Set the stir plate to mix at l OOrpm. Label this Beaker with "3^ld Exchange". Incubate these dialysis tubes for four hours at 4°C.

Slowly remove the dialysis tubes from the 5 liter beaker and place them in the other 5 liter beaker of I xPBS pH 7.4 that has been stored in the 4°C. Make sure not to mix or agitate the solution inside of the dialysis tube. There may be some precipitation at this point. Place a magnetic stir bar in the beaker. Set the stir plate to mix at l OOrpm. Label this Beaker with "4^th Exchange". Incubate these dialysis tubes for four hours at 4°C.

Hook up a 1 Liter Filter System bottle to a vacuum pump. Remove the 5 liter beaker that contains the dialysis tubes from the refrigerator. One at a time, carefully empty the contents of the dialysis tube into the top of the filter system. Once all the dialysis tubes have been emptied into the top reservoir of the filter make sure to save a 50μ1 aliquot of the solution for gel analysis. Label the aliquot "rGST-I2 dialyzed pre- filtered" and store it at 4°C until needed.

Filter the solution into the 1 Liter System bottle. Once filtering is complete save a 50μ1 aliquot of the solution for gel analysis. Label the aliquot "rGST-I2 dialyzed filtered" and store it at 4°C until needed. The volume of the filtered solution should be approximately 550mls after dialysis. Label the bottle with "rGST-I2 dialyzed filtered-l ^st agarose incubation" and today's date. Place this bottle in the 4°C while the immobilized glutathione agarose is being prepared.

Add immobilized glutathione agarose to the bottle of rGST-I2 as follows in steps 5.5.7 through 5.5.10 - all steps.

Measure and equilibrate the immobilized glutathione agarose (resin). Take a 20ml chromatography column and place it in a clamp attached to a ring stand. Place a 250ml beaker under the column to act as a waste basin.

Take the bottle of pre-made agarose and mix it well until the agarose is suspended evenly in the storage solution.

Using a 10ml serological pipet load this mixed agarose into the column. Continue to load the mixture until a 6ml bed of the agarose has settled at the bottom of the column. Let the solution in the column drain out until the top surface of the storage solution is 1 ml above the agarose bed. At that point cap the bottom of the column. (While working with this agarose do not let it dry out. Keep it wet at all times.)

5.5.10.1. Uncap the column. Wash the agarose with 60ml of degassed l xPBS pH 7.4. Cap the column.

5.5.10.2. Remove the 1 liter bottle containing the ~550ml of rGST-I2 solution from the refrigerator.

5.5.10.3. Load 10ml of degassed l xPBS pH 7.4 into the column.

Using a 10ml serological pi pet, suspend the agarose thoroughly and add it to the 1 liter bottle of rGST-I2 solution.

5.5.10.4. Add 10ml of degassed l xPBS pH 7.4 into the column. Mix this 10ml volume to retrieve the residual agarose that is stuck to the column. Add that 10ml volume to the 1 liter bottle of rGST-I2 solution.

5.5.10.5. Cap the bottle and slowly rotate the bottle by hand to mix the agarose thoroughly into solution.

5.5.10.6. Place the bottle in the 4°C refrigerator and incubate it

overnight.

Run a protein gel to determine if the GST-I2 has been processed properly up to the point where the GST-I2 is filtered in l xPBS pH 7.4. The following samples are run on this gel:

5.6.2.1 . Denatured GST-I2-, Step 5.3.47. - DEN

5.6.2.2. Diluted refolded GST-I2, Step 5.4.7. - DIL

5.6.2.3. Pre-filtered rGST-I2 in 1 xPBS pH7.4, Step 5.5.3. - PRE

5.6.2.4. Filtered rGST-I2, Step 5.5.4. - FIL

Sample preparation: Label four micro centrifuge tubes with DEN, DIL, PRE and FIL.

To prepare the DEN sample, add the following to the tube:

5.6.4.1. Ι μΐ of Sample

5.6.4.2. ΙΟμΙ of 4X Sample Buffer

5.6.4.3. Ι μΐ of 2.5% beta-mercaptoethanol

5.6.4.4. 28μ1 of Nanopure distilled H₂0 To prepare the DIL, PRE and FIL samples, add the following to each tube:

5.6.5.1. 20μ1 of Sample

5.6.5.2. Ι ΟμΙ of 4X Sample Buffer

5.6.5.3. Ι μΐ of 2.5% beta-mercaptoethanol

5.6.5.4. 9μ1 of Nanopure distilled H₂0

Decant the Simply Blue Safe Stain and add 100ml of Nanopure distilled FLO. Place the gel back on a rocker and incubate it for 1 hour. Within l Omins of this incubation step you be able to confirm that the GST-I2 antigen is present in the filtered sample (FIL) (Figure 4C). When the bands are confirmed move on to the next step.

First round of purification of the rGST-I2 antigen.

Set up two columns on a ring stand. Label each column with either #1 or #2. Under each column place a 250ml beaker to act as a waste container. Snap the seal on the bottom of the column to open up the column. Pre-rinse each column with 50ml of Nanopure Distilled H₂0. Place a 1 L disposable sterile bottle below each column. Pull the "rGST-I2 dialyzed filtered- 1 ^st agarose incubation" bottle out of the refrigerator and slowly mix the bottle.

The mixture inside of the bottle will now be evenly split into two separate columns. The solution is split into two columns because larger volume beds decrease the flow rate. Splitting the column work will allow this purification procedure to be performed in approximately two hours.

Using a 25ml serological pipet, begin to load each column with the agarose suspension. Fill the column to the top with the suspension. Then let the volume drop to the 15ml mark on the column, at that point fill the column to the top again. You do not want to let the volume of the agarose suspension drop too low. If that occurs, the addition of your next agarose suspension could disturb the formation of the agarose bed. Make sure you are adding the suspension slowly to reduce the disturbance to the forming agarose bed.

Continue loading the column with the agarose suspension. When all of the agarose suspension is loaded on the column it should be capped. Cap them when the liquid phase in the column reaches the 10ml mark on the column. Each bed volume will be approximately 3mls in size. Take this time to prepare I xPBS pH 7.4 ( 1 liter). Degas the PBS with argon for 5 minutes before using.

At this point remove each 1 liter bottle from underneath the column. Combine the flow through volumes of each bottle into one bottle. Label this bottle "rGST-I2 dialyzed filtered-2^nd agarose incubation" with today's date and store it in the refrigerator until it is needed. Place 250ml Beakers under each column to act as waste containers. Begin washing the bed with 60ml of degassed I xPBS pH 7.4 in 2x30ml increments to each column. The PBS should be degassed for 5mins with argon immediately before it is used. Be sure to load the wash buffer very slowly.

After the last wash is added, let the volume drop till the meniscus is 1 - 2mm above the bed. Cap the column at this point. You will now degas the elution buffer ( I xPBS pH 7.4 w/lOOmM reduced

glutathione). This elution buffer is degassed with argon for 2 minutes before using.

Load 1.5 ml of elution buffer to each capped column and let it stand for 5 minutes. Uncap the columns and collect the elutions in a 1.5ml micro centrifuge tube. Collect the elution until the volume reaches approximately 1.2mls. Cap the columns. These are the first elutions. Label each tube with "Elution#, column#" and today's date.

Monitoring of the elutions is done in parallel of this elution procedure using Bradford reagent. 5ul of each elution is added to separate wells on a 96 well plate. 250ul of Bradford reagent is then added to each well. The presence of protein will be identified in this procedure by the changing of the reagent color from brown to blue. If the elution gives off a blue color on the Bradford mark the respective micro centrifuge tube with a "B".

Repeat steps 5.6.17. through 5.6.18.three more times to collect elutions 2-4. At this point you should see the blue color of elution #4 disappearing on the 96-well plate. You will have a total of 8 micro centrifuge tubes at this point.

Store the elutions at 4°C. They will be used later when the elutions are pooled.

Wash each resin bed with 80ml of degassed I xPBS pH 7.4.

Using a 10 ml serological pipet suspend each bed in 10ml of I xPBS pH 7.4 and added it to the bottle labeled "rGST-I2 dialyzed filtered-2^nd agarose incubation".

Add an additional 10ml of degassed I xPBS pH 7.4 to each column and mix the solution well to retrieve any residual agarose. Add this volume to the bottle labeled "rGST-I2 dialyzed filtered-2^nd agarose incubation". Rotate this bottle slowly to mix the resin. Store the bottle overnight at 4°C.

DAY 7

5.7.1. Repeat steps 5.6.9. through 5.6.23. This will generate a flow through bottle labeled as "rGST-I2 dialyzed filtered-3^ld agarose incubation" and produce a new set of 8 elutions that are stored overnight at 4°C. DAY 8

5.8.1 . Repeat steps 5.6.9. through 5.6.23. This will generate a bottle labeled as "rGST-I2 dialyzed filtered-final flow through" and produce a new set of 8 elutions that are stored at 4°C.

5.8.2. Pool the elutions that were marked with a "B" into one final volume.

Only pool the elutions that show a blue color on the Bradford assay.

5.8.2.1. Spin the elution tubes in a micro tube centrifuge for 1

minute at 5000xg to remove any precipitants.

5.8.2.2. Combine the elutions into one 15ml conical tube. Label that tube "rGST-I2 pooled elutions".

5.8.3. Perform a Bradford Assay to determine the concentration of the protein using the Bradford Dye and a pre-made albumin standard.

5.8.3.1 . Standard A comes in a sealed ampoule and acts as you stock solution. The standard diluent is lxPBS pH 7.4 w/ l OOmM reduced glutathione.

Follow the table below for setting up the standard curve.

F 325 μΐ 325 μΐ of vial Ε dilution 250 μg/ml

G 325 μΐ 325 μΐ of vial F dilution 125 μg/ml

H 400 μΐ 100 μΐ of vial G dilution 25 μg/ml

I 400 μΐ 0 0 μg/ml = Blank

5.8.3.3. Load 5ul of the standards and the pooled rGST-I2 sample into a 96 well plate. Load them in duplicate.

5.8.3.4. Add 250ul of Bradford reagent into each well. Gently tap the plate to mix the samples. Incubate the plate for 5mins then read the plate at 595nm on micro plate reader. Do not over incubate the plate.

5.8.3.5. Graph the data on Excel. Graph the absorbance at 595 nm

(x-axis) vs. concentration g/ml (y-axis). Only graph from the range of 1 ,000 g/ml through 25 g/ml, because this is the linear part of the curve. Through linear regression determine the formula of the curve. It will be in y=mx+b format. Use this formula to determine the concentration of your sample.

Once the concentration has been determined, aliquot the antigen and freeze it in liquid nitrogen. Label the aliquots. Then store the antigen at -70. Save an aliquot for gel analysis.

Run a gel to determine that the GST-I2 has been purified.

5.8.5.1. Sample preparation: Label a micro centrifuge tubes with refolded GST-I2. Add the following to the tube. Load 2ug of protein in the well. X=2.7ug of GST-I2, where 30μ1 of sample prep will be loaded from a total sample prep volume of 40μ1

5.8.5.1.1. Χμΐ of Sample

5.8.5.1.2. Ι ΟμΙ of 4X Sample Buffer

5.8.5.1.3. Ι μΐ of 2.5% beta-mercaptoethanol

5.8.5.1.4. 29-X μΐ of Nanopure distilled H₂0

.8.6. Mix the samples and then incubate them in a heat block at 90°C for 5 minutes. Repeat steps 5.3.5. through 5.3.7. to prepare and process the gel.

.8.7. Decant the Simply Blue Safe Stain and add 100ml of Nanopure

distilled H₂0. Place the gel back on a rocker and incubate it for 1 hour. 6.0 QUALITY CONTROL

[0325] Each lot of purified rGST-I2 antigen is compared to two previous lots to ensure the reproducibility of purification.

7.0 ANALYSIS

[0326] Each lot of purified rGST-I2 antigen is compared to BSA standards to determine the concentration using the Bradford Assay and linear regression.

8.0 REFERENCES

[0327] "Purification and characterization of recombinant extraxellular domain of human HER2 from Escherichia coli" Protein Expression and Purification X. Liu et al. 2007 pages 247-254.

Example 10. Protocol for Performing Anti-I2 Immunoassays.

1.0 PURPOSE

[0328] This anti-I2 Indirect Sandwich ELISA procedure details the steps necessary for the quantitative determination of Human IgA serum antibodies against 12.

2.0 SCOPE

[0329] The 12 Immunoassay test is used to detect serum concentrations of anti-I2 in patient samples.

3.0 PRINCIPLE

[0330] The assay employs an indirect sandwich immunoassay format where capture antibodies are coated on the bottoms of the wells of a 96-well microplate. The plate is then blocked to minimize non-specific binding and high background. Antigen is added to the plate which binds to the capture antibody. Excess antigen is washed away after incubation.

[0331] The calibrators, controls, and patient samples are incubated in the appropriate wells and the biomarker binds to the antigen. Unbound biomarker is then washed away and the detection antibody labeled with alkaline phosphatase is incubated in the wells. The plate is washed again and a chemiluminescent substrate solution is added. The plate is read on Molecular Device' s Spectramax M5^e using luminescent detection.

4.0 DEFINITIONS

4.1. 12: Pseudomonas fluorescens-related peptide

4.2. ELISA: Enzyme-linked immunosorbant assay 5.0 SAMPLE REQUIREMENTS

[0332] Patient's whole blood is drawn into Serum Separator Tube (SST) and

EDT A/Lavender Top tube. The tubes are shipped within 7 days to Prometheus Laboratories, under room temperature conditions or using Cold pack. Prior to shipment, the tubes are stored under refrigerated conditions.

6.0 PROCEDURE

6.1. Prepare coating buffer by diluting 20X PBS to I X with Nanopure Water.

6.2. Dilute Mouse a-GST mAb to 5 g/mL in 1 X PBS. Coat plates at

ΙΟΟμίΛνεΙΙ. Store overnight at 4°C.

6.3. Add 5% Mouse Serum to a volume of 12 Dilution Buffer (1 XPBS+1 % BSA+0.5% PVA + 0.8% PVP) needed for 1 ° & 2° dilutions for the day to create 12 Working Buffer, [e.g., 2 mL Mouse Serum to 38 mL 12 Dilution Buffer]

6.4. Bring plates, Histidine Blocking Buffer (20mM Histidine + 0.5M NaCl-f- 1 %BS A) and 12 Working Buffer to room temperature prior to use. All other reagents, controls, standards, and samples should be kept on ice or at 4°C prior to use.

6.5. Prepare Standard Curve by diluting Anti-His-I2 Rabbit Serum in 12

Working Buffer; standard curve is plated in duplicate.

Su ested dilutions for Standard Curve (2 plates):

6.6. Prepare wash buffer by diluting 20X PBS-Tween to IX with DIH₂0.

6.7. Wash wells 3 times with 300μίΛνε11 I X PBS-Tween.

6.8. During blocking step, prepare dilution of each positive Control to be

assayed; [e.g., 25μL· Control sample into 225μί] of 12 Working Buffer for a 1 : 10 dilution; Negative control is a 1 : 100 dilution [e.g., 2.5μΙ, Stripped Serum into 250μί Diluent] ; all Control samples are plated in duplicate. Samples will be diluted 1 : 100 & 1 :200 [e.g., ΙΟμί sample + 90μί 12 Working Buffer for 1 : 10; 25 μΐ, of 1 : 10 into 225μί 12 Working Buffer for 1 : 100; \ 5\iL of 1 : 10 into 285μΙ_ 12 Working Buffer for 1 :200] . All samples are plated in duplicate.

NOTE: Incubate diluted samples and standards on the bench for the duration of the antigen step.

6.9. Block wells with 300 μίΛ εΙΙ of Histidine Blocking Buffer. Incubate for 1 hour at room temperature with shaking (approx 300 rpm).

6.10. Dump blocking solution. Do not wash plate.

6.1 1. Dilute rGST-I2 in IX PBS (according to the formula below) immediately before coating on plates @ 5μg/mL, ΙΟΟμίΛνεΙΙ. [V_F = total volume needed; i.e., 1 1 mL total volume = one plate]

Formula: Initial Concentration of Antigen Stock (ug/mL) = Dp (Dilution Factor) = I_c/F_c

5 g/mL (final desired concentration)

V_F/D_F = volume of Stock (V_s) needed to add to 1 XPBS

V_F- (Vp/Dp) = Volume of IX PBS (V_P); V_s + V_P = V_F @ 5μg/mL cone, of Antigen e.g., If Antigen Stock is 1788 μg/mL, then 1788μg ml / 5μg/mL = 357.6 (D_F); for one plate V_F = l l mL. 1 l mL/357.6 = 0.031 mL of Stock antigen (i.e., 31 μΐ.)

1 1 mL - 0.031 mL = 10.969 mL of I X PBS

0.031 mL Antigen Stock + 10.969 mL of IX PBS = 1 1 mL @ 5 g/mL

Incubate 1 hour with shaking.

6.12. Wash wells 3 times with 300μίΛνε11 of lXPBS-Tween.

6.13. Dilute Tropix Assay Buffer (10X) to IX with DDIH₂0 for use in step

10.19. Keep buffer at 4°C prior to use and use cold.

6.14. Add 100

calibrators and samples to plate in duplicate. Incubate 1 hour at room temperature on orbital shaker (approx. 300-700 rpm).

6.15. Wash wells 3 times with 300μίΛνε11 of lXPBS-Tween.

6.16. Add 100 ίΛ βΙΙ of 1 :5,000 secondary antibody diluted in 12 Working Buffer, [e.g., 1.2 iL Goat anti-Rabbit IgG to 6 mL 12 Working Buffer for Standard Curve; 2\iL Goat anti-Human IgA to lOmL 12 Working Buffer for all patient and control samples.] Incubate 1 hour at room temperature with shaking.

6.17. Wash wells 3 times with 300μL/well of lXPBS-Tween.

6.18. Wash wells 2 times with 200μΕΛνε11 Tropix Assay Buffer ( I X). 6.19. Add 100

of the chemiluminescent substrate solution [l OmL for one plate]. Substrate should be kept at 4°C prior to use and used cold. Incubate for 20 minutes, protected from light - with shaking.

6.20. Immediately read plates on Spectramax M5^e using luminescence protocol, top read, opaque 96 well plate, with Integration set at 500.

7.0 QUALITY CONTROL

7.1. The Blank for each plate is determined by graphing of the standard curve.

7.2. The High, Medium, and Low Control values generated in the assay may be evaluated.

7.3. Figure 5 shows a graph of a sample standard curve with controls. Standard and control data are evaluated and graphed using Softmax.

8.0 ANALYSIS

8.1. The assay is measured in EU.

8.1.1. Reference Range is 367.80 EU. Samples with values greater than

367.80 EU will be considered positive for anti-I2.

8.1.2. Minimum Detectable Concentration (MDC) is 1.81 EU.

8.1.3. Reportable Range is 2.5 EU - 100 EU.

8.1.4. Patient value less than 2.5 EU will be reported as < 2.5 EU.

8.1.5. Patient value that exceeds 100 EU will be reported as > 100 EU.

8.2. Testing must be repeated on samples with >15% CV between duplicates or if both duplicates are below the lower limit of the reportable range.

9.0 CALIBRATION

[0333] A 7-point calibration curve is run with each assay and must meet expected criteria; each curve is compared to a reference set from 30 previous assays in order to determine acceptability.

10.0 LINEARITY

[0334] Assay linearity and reportable range are verified semiannually using the appropriate testing materials and statistical analysis.

11.0 INTERFERENCE

[0335] This assay was tested for interference by Rheumatoid Factor, hemolysis and various substances (Bilirubin (400 ug/mL), Cholesterol (5 mg/mL), Heparin (80 U/mL), EDTA (1.8 mg/mL) and Hemoglobin (5 mg/mL). Anti-I2 detection was found within acceptable range following spiking with all of these substances.

Example 11. Protocol for Validating Anti-I2 Immunoassays.

[0336] This example provides a protocol for the validation of human anti-I2 ELISA.

A. Reference Range

[0337] The reference range will be done by one analyst performing the assay on one day (two plates). Forty healthy control samples will be tested in duplicate. The reference range will be determined from anti-I2 concentration. Mean value, standard deviation, minimum value and maximum value will be calculated. 95% Confidence intervals (mean ± 1 .96 standard deviation) will be considered as the normal range.

B. Validation

[0338] Performance of the assay will be done by 3 analysts performing the assay on five different days (total 15 assays). The validation will be performed using 3 lots antigen preparation. The study will distinguish operator and batch effects. Each of the three operators will use a different lot at least one time during the five days validation.

B.l. Standard Curve

[0339] The curve will be derived from 7 standards that range from 1 :2000 to 1 :320,000 dilutions and a blank. Serial dilution will be performed from a 1 :500 stock. The stock 1 :500 dilution will be prepared by adding 2 μΐ of anti-His rabbit serum to 998 μΐ assay diluent. To make the initial 1 :2000 dilution, 163 μΐ of the stock will be added to a tube containing 489 μΐ of assay diluent. Subsequent dilution will be performed as described in the table below.

Suggested dilutions for Standard Curve (2 plates)

[0340] Each standard will be assayed in duplicate. The reproducibility of the standard curve will be assessed by comparing for each lot the Expected value with the Mean Observed/Calculated, Standard Deviation and %CV. The analysis will show pair-wise comparison between multiple standard lots. Acceptable signal reproducibility for standard 1 - 7 will be defined as precision (%CV) less than 10%.

B.2. Sensitivity

[0341] The minimum detectable concentration (MDC) will be determined using a total of 20 replicates of the zero standards (blank). The Mean and Standard deviation will be used to calculate the MDC. MDC will be determined by adding two standard deviations to the mean optical density value of the 20 zero standard replicates.

B.3. Precision/Accuracy

[0342] The intra and inter-assay precision will be determined for high, medium and low positive controls. For intra-assay precision (precision within the assay), high, medium and positive controls will be tested in replicates of 16 on a single plate. For inter-assay precision (precision between assay), high, medium and positive controls will be tested in fifteen separate plates. Each sample will be assessed for each run. Assigned values, Mean, Standard Deviation and %CV will be calculated. Acceptable analytical precision for samples spanning the standard curve dynamic range will be defined as precision (%CV) less than 10%.

B.4. Reportable Range/Linearity

[0343] The dilution linearity will be evaluated using five serial two-fold dilution of the high positive, medium or low controls (Neat), starting from ½. Each will be assessed in duplicate. Yield of anti-I2 concentration will be obtained when multiplied by the dilution factor.

Percent of recovery will be determined. Performance will be considered acceptable when the results are between 80% and 120% of the expected concentration. Linear regression (R²) will be calculated to confirm that the sample dilution correlate linearly with the calculated ELISA units.

C. Stability Studies

[0344] Stability assays will be performed by 3 analysts the same day (3 plates). Each sample assay will be previously prepared and stored at -80 °C.

C.l. Room temperature stability (RT)

[0345] High, Medium and Low controls will be incubated at room temperature for 1 , 2, 4 or 7 days. The treated controls will be assayed and compared to the non-treated controls.

Acceptable criteria: 80- 120% of initial calculated 12 concentration. C.2. 4°C Temperature stability (4°C)

[0346] High, Medium and Low controls will be incubated at 2-8°C for 1 , 2, 4 or 7 days. The treated controls will be assayed and compared to the non-treated controls. Acceptable criteria: 80- 120% of initial calculated anti-I2 concentration.

C.3. Freeze & Thaw (F/T) purified GST-I2 antigen preparation and samples

[0347] High, Medium and Low controls will be subjected to 5 freeze and thaw cycles. The treated controls will be assayed and compared to the non-treated controls. Acceptable criteria: 80- 120% of zero freeze-thaw.

[0348] Aliquots of GST-I2 antigen will be subjected to 1 -5 cycles (I2-FT0.1 ,2,3,4,5) of freeze-thaw and will be assayed and compared with samples kept frozen. Acceptable criteria: 80-120% of zero freeze-thaw

C.4. Standard stability

[0349] For standard stability evaluation, standard stock solution will be divided into two aliquots and stored at 4°C for 7 days and 14 days. The assay will be performed using high, medium and low controls. Acceptable criteria: 80-120% of zero freeze-thaw.

D. Interference/Specificity

[0350] Interference assays will be performed by 3 analysts the same day (3 plates). D.l. Hemolyzed serum

[0351] Hemolysed serum will be tested for anti-I2 assay interference. Whole blood will be collected from three healthy consented donors. The blood will be vortexed vigorously to cause severe hemolysis and then allowed to clot. Serum will be collected. High, Medium and Low controls will be diluted in duplicate with an equal volume of NHS or Hemolysed normal sample. Acceptable criteria: 80-120% of initial calculated anti-I2concentration.

D.2. RF serum

[0352] To determine if Rheumatoid Factor (RF) will interfere with the assay, High, Medium and Low controls will be diluted in duplicate with an equal volume of normal human Serum (NHS) or Rheumatoid factor (RF) positive serum (clinical sample purchased from Aalto Scientific). Anti-I2 recovery from controls spike with NHS will be compared with controls spiked with RF positive serum. Acceptable criteria: 80-120% of initial calculated anti-I2 concentration.

D.3. Specificity [0353] The effect of various substances on the performance of anti-I2 assay will be determined. High, medium and low controls will be spiked with Bilirubin (400 ug/mL), Cholesterol (5 mg/mL), Heparin (80 U/mL), EDTA ( 1 .8 mg/mL) and Hemoglobin (5 mg/mL). % 12 recovered in the spiked control will be calculated. Acceptable criteria: 80- 120% of initial calculated anti-I2 concentration.

Example 12. Exemplary Anti-I2 Immunoassays Using Refolded GST-I2 Antigen.

[0354] This example describes two anti-I2 immunoassays which utilize refolded GST-I2 antigen (see, Example 9) to detect anti-I2 antibodies in a biological sample. Both assays are performed on a 96- well microtiter plate with a refolded GST-tagged protein consisting of 100 amino acids of the 12 sequence. However, one of ordinary skill in the art will appreciate that a fragment of the 12 polypeptide that is immunoreactive with an anti-I2 antibody is suitable for use in the immunoassays described herein.

[0355] In one embodiment, the anti-I2 assay is the ELISA depicted in Figure 6A and described in Example 10. In particular, refolded GST-I2 antigen is captured on the plate using a monoclonal anti-GST antibody coated on the well surface. After incubation of patient serum samples in the wells, detection of anti-I2 IgA/IgG is accomplished using an alkaline phosphatase enzyme-conjugated anti-human IgA/G reagent. The reaction is then revealed using a cheminulescent substrate solution.

[0356] To assess the prognostic value of this assay, anti-I2 serum values were analyzed for patients with CD complications (e.g., penetrating or fibrostenosing) and CD patients having undergone a surgical procedure. The results showed that 64.6% of patients with high levels of anti-I2 (e.g., levels above a reference concentration level) experienced complicated disease behavior, compared to 52.2% of patients with low levels of anti-I2 (p = 0.002). As such, the detection of anti-I2 using this robust assay finds utility in predicting possible disease behavior outcomes for CD patients.

[0357] In another embodiment, the anti-I2 assay is the ELISA depicted in Figure 6B. In particular, the plate was coated with 100 μΐ/well of neutravidin in sodium carbonate buffer pH 9.5 at 4°C overnight. After washing with PBST, the plate was blocked with SuperBlock for 30 minutes. After washing with PBST, half of the plate was incubated with 100 μΐ of biotinylated refolded GST-I2 (Bio-GST-I2; 100 μ^πιΐ in SuperBlock), while the other half was incubated with 100 μΐ of SuperBlock (background) for 1 hour at room temperature (RT) with gentle agitation. Pooled IBD patient serum was used as a standard. The arbitrary unit of the standards was set as 160 U/ml for IgA GST-I2 and 146 U/ml for IgG GST-I2. Serial dilutions of the standard were made to generate the standard curve (3 U/ml and then 1 :3 dilutions). 100 μΐ/well of the standards and samples (1 :300 dilution in SuperBlock) were added to each well after washing with PBST. After incubating at RT for 1 .5 hours with gentle agitation, the plate was washed and incubated with 100 μΐ of HRP-labeled anti-human IgA or IgG 2° antibody for 1 hour at RT with agitation. TMB substrate was added to each well after washing. The plate was incubated in the dark with agitation for 15 minutes and the reaction was stopped with 50 μΐ/well of 1M phosphoric acid. A SpectraMax plate reader was used to read the OD450. To analyze specific binding, the background OD450 from standards and samples were subtracted from the corresponding OD450 from Bio-GST-I2-containing wells. The values of IgA or IgG GST-I2 were calculated from the standard curve using the Prism graphPad program.

[0358] It is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of skill in the art upon reading the above description. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references, including patent applications, patents, PCT publications, and Genbank Accession Nos., are incorporated herein by reference for all purposes.

Claims

\\ 1 1 _ί V 1 H3 V 1 _> A 1 VI 1.1 u; 1. A method for predicting a risk of developing dysplasia/cancer and/or an inflammatory complication in an individual following a surgical procedure whereby the colon is removed and an internal pouch is created, said method comprising:

(a) analyzing a sample obtained from said individual to determine the presence, level, or genotype of one or more markers in said sample to obtain a marker profile; and

(b) predicting a risk that said individual will develop dysplasia/cancer and/or an inflammatory complication following said surgical procedure based upon said marker profile.

2. The method of claim 1, wherein said individual is diagnosed with ulcerative colitis (UC) or indeterminate colitis (IC) prior to said surgical procedure.

3. The method of claim 1 or 2, wherein said surgical procedure is an ileal pouch-anal anastomosis (IPAA) procedure.

4. The method of any one of claims 1 to 3, wherein said dysplasia/cancer is cancer of the pouch.

5. The method of any one of claims 1 to 4, wherein said inflammatory complication is selected from the group consisting of pouchitis, Crohn's disease of the pouch, cuffitis, pouch stricture, pouch sinus, proximal small bowel bacterial overgrowth, irritable pouch syndrome (IPS), anismus, and a combination thereof.

6. The method of any one of claims 1 to 5, wherein said one or more markers is selected from the group consisting of a serological marker, a genetic marker, and a combination thereof.

7. The method of claim 6, wherein said serological marker is selected from the group consisting of an anti-neutrophil antibody, an anti-Saccharomyces cerevisiae antibody, an antimicrobial antibody, an acute phase protein, an apolipoprotein, a defensin, a growth factor, a cytokine, a cadherin, and a combination thereof.

8. The method of claim 7, wherein said anti-neutrophil antibody is selected from the group consisting of an anti-neutrophil cytoplasmic antibody (ANCA), perinuclear anti-neutrophil cytoplasmic antibody (pANCA), and a combination thereof.

9. The method of claim 7, wherein said anti-Saccharomyces cerevisiae antibody is selected from the group consisting of anti-Saccharomyces cerevisiae

immunoglobulin A (ASCA-IgA), anti-Saccharomyces cerevisiae immunoglobulin G (ASCA- IgG), and a combination thereof.

10. The method of claim 7, wherein said antimicrobial antibody is selected from the group consisting of an anti-outer membrane protein C (anti-OmpC) antibody, an anti-I2 antibody, an anti-flagellin antibody, and a combination thereof.

11. The method of claim 6, wherein said serological marker is selected from the group consisting of ASCA-IgA, ASCA-IgG, anti-OmpC antibody, anti-CBir- 1 antibody, anti-I2 antibody, ANCA, pANCA, and a combination thereof.

12. The method of claim 6, wherein the presence or level of said serological marker is detected with a hybridization assay, amplification-based assay, immunoassay, or immunohistochemical assay.

13. The method of claim 6, wherein said genetic marker is at least one of the genes set forth in Tables 1 A- IE.

14. The method of claim 6, wherein the genotype of said genetic marker is detected by genotyping for the presence or absence of a single nucleotide polymorphism (SNP) in said genetic marker.

15. The method of claim 14, wherein said SNP is at least one of the SNPs set forth in Tables I B- IE.

16. The method of claim 14, wherein said genetic marker is NOD2 and said SNP is selected from the group consisting of SNP8, SNP12, SNP13, and a combination thereof.

17. The method of any one of claims 1 to 16, wherein said marker profile is determined by detecting the presence, level, or genotype of at least two, three, four, five, six, seven, eight, nine, or ten markers.

18. The method of any one of claims 1 to 17, wherein said sample is selected from the group consisting of serum, plasma, whole blood, and stool.

19. The method of any one of claims 1 to 18, wherein said sample is obtained from said individual prior to said surgical procedure.

20. The method of any one of claims 1 to 19, wherein step (b) comprises comparing the level or genotype of each of said markers in said marker profile to a reference level or genotype to predict a risk that said individual will develop dysplasia/cancer and/or an inflammatory complication following said surgical procedure.

21. The method of any one of claims 1 to 20, wherein step (b) comprises applying a statistical analysis to said marker profile to predict a risk that said individual will develop dysplasia/cancer and/or an inflammatory complication following said surgical procedure.

22. The method of claim 21, wherein said statistical analysis is a quartile analysis.

23. The method of claim 22, wherein said quartile analysis converts the presence, level or genotype of said one or more markers into a quartile score.

24. The method of claim 23, wherein said marker profile is a quartile sum score (QSS) for said individual obtained by summing said quartile score for each of said one or more markers.

25. The method of claim 21, wherein said statistical analysis is a median analysis.

26. The method of any one of claims 1 to 25, wherein said marker profile predicts that said individual has a high risk of developing dysplasia/cancer following said surgical procedure.

27. The method of any one of claims 1 to 26, wherein said marker profile predicts that said individual has a high risk of developing an inflammatory complication following said surgical procedure.

28. The method of any one of claims 1 to 25 and 27, wherein said marker profile predicts that said individual has a low risk of developing dysplasia/cancer following said surgical procedure.

29. The method of any one of claims 1 to 26 and 28, wherein said marker profile predicts that said individual has a low risk of developing an inflammatory complication following said surgical procedure.

30. The method of any one of claims 1 to 29, further comprising recommending a course of therapy for said individual based upon said prediction.

31. The method of claim 30, wherein said course of therapy is selected from the group consisting of an anticancer drug, an IBD therapeutic agent, and a combination thereof.