This invention is related to the testing of cytokines for diagnostics, disease monitoring, and therapy monitoring. In particular, it relates to Irritable Bowel Syndrome.
Irritable bowel syndrome (IBS) is one of the most common functional gastrointestinal disorders, characterized by abdominal pain or discomfort/bloating and associated with disturbances in abnormal bowel function (diarrhea and/or constipation). Although only a minority of sufferers seek care for their symptoms, IBS accounts for annual direct costs of $8 billion and indirect costs of $25 billion in the United States, and an prevalence of 10-15% in N. America. Despite the prevalence and impact of IBS in the community, the pathophysiology of IBS is not fully understood, which in part is due to the fact that the etiology is not explainable by obvious local biochemical or structural causes. The disorder is now attributed to dysregulation of the brain-gut axis, with alterations at different levels and disturbed interplay of several factors, specifically the enteric, autonomic and/or central nervous systems, mucosal immune activation, an altered microbiotome, and psychosocial factors.
The therapeutic strategy for IBS has been focused on traditional therapies focused on individual symptoms, with limited efficacy in addressing the entire syndrome complex without any effects on the natural history of the disorder. The lack of definitive biomarkers has hampered diagnosis, prognosis, and the development of fully effective treatments.
The hallmark symptoms of IBS are similar to that of other gastrointestinal diseases, including Inflammatory Bowel Disease (IBD), which is characterized by chronic, progressive, systemic, autoimmune inflammatory disorder of the gastrointestinal tract. Some studies have also suggested that IBS is a type of low-grade IBD, and that these diseases may be inter-related. The diseases are however clinically distinct because IBS does not produce the destructive inflammation or intestinal bleeding found in IBD, or the harmful complications often occurring with IBD. Patients with IBS are not at higher risk for colon cancer, nor are they more likely to develop IBD or other gastrointestinal diseases. Furthermore, IBS seldom requires hospitalization, and treatment does not usually involve surgery or powerful medications, such as steroids or immunosuppressive agents. There is, however, similarity in early symptoms and signs of IBS with IBD, and given the lack of definitive biomarkers to distinguish between these two disorders, clinicians are often faced with difficult challenges to identify and distinguish between these two clinically distinct diseases.
Cytokines are defined as any of several regulatory proteins, such as the interleukins and lymphokines, that are released by cells of the immune system and act as intercellular mediators in the generation of an immune response. Cytokines are secreted by immune or other cells, whose action are on cells of the immune system, such as, but not limited to, T-cells, B-cells, NK cells and macrophages. Chemokines are defined as chemotactic cytokines produced by a variety of cell types in acute and chronic inflammation that mobilize and activate white blood cells. Cytokines and chemokines are important cell signaling proteins, mediating a wide range of physiological and pathological responses, including immunity, inflammation, and hematopoiesis.
Several therapeutic agents, primarily directed at cytokines, are currently available and have shown great promise in the treatment of various inflammatory conditions of the bowel. While previous studies have evaluated systemic cytokine profiles in IBS, they have been limited to a relatively small number of cytokines and the analysis of absolute level of each cytokine, without consideration of the interplay of multiple cytokines. There are no tests available for determining whether patients have a specific cytokine antagonized by a therapeutic agent, or whether patients will positively respond to such medications. Furthermore, despite the need to identify cytokine associations with IBS, there have been no definitive link identified between cytokine levels and diagnosis, prognosis, and treatment response in such pathologic states. There is a continuing need in the art better to identify and distinguish IBS and to monitor the course of the disease.
According to one aspect of the invention a method is provided to aid in diagnosing Irritable Bowel Syndrome (IBS). A patient sample is tested to determine level of one or more cytokines selected from the group consisting of IL-5, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17, and CCL-2. The patient level is compared to a reference range of levels determined in healthy subjects. A level in the patient that falls outside of the reference range is identified as indicating IBS.
Another aspect of the invention is a method to aid in distinguishing between IBS and Irritable Bowel Disease (IBD). A patient sample is tested to determine level of one or more cytokines selected from the group consisting of IL-6, IL-10, IL-12, TNF-α, and CCL-2. The patient level is compared to a reference range of levels determined for IBS patients and to a reference range of levels determined for IBD subjects. A level in the patient that falls within the IBS reference range is identified as indicating IBS and a level in the patient that falls within the IBD range is identified as indicating IBD.
Still another aspect of the invention is a method to monitor response to a therapy for IBS in a patient receiving therapy. A patient sample is tested to determine level of one or more cytokines selected from the group consisting of IL-1β, IL-6, IL-12, TNF-α, and CCL-2. The level is compared to a reference level previously determined in the patient prior to therapy or at a previous time point during therapy. A change in the level compared to the reference is identified as indicating responsiveness to the therapy.
According to one aspect of the invention a method is provided to aid in diagnosing Irritable Bowel Syndrome (IBS). A patient sample is tested to determine level of one or more mRNA molecules encoding a cytokine selected from the group consisting of IL-5, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17, and CCL-2. The patient level is compared to a reference range of levels determined in healthy subjects. A level in the patient that falls outside of the reference range is identified as indicating IBS.
Another aspect of the invention is a method to aid in distinguishing between IBS and Irritable Bowel Disease (IBD). A patient sample is tested to determine level of one or more mRNA molecules encoding a cytokine selected from the group consisting of IL-6, IL-10, IL-12, TNF-α, and CCL-2. The patient level is compared to a reference range of levels determined for IBS patients and to a reference range of levels determined for IBD subjects. A level in the patient that falls within the IBS reference range is identified as indicating IBS and a level in the patient that falls within the IBD range is identified as indicating IBD.
Still another aspect of the invention is a method to monitor response to a therapy for IBS in a patient receiving therapy. A patient sample is tested to determine level of one or more mRNA molecules encoding a cytokine selected from the group consisting of IL-1β, IL-6, IL-12, TNF-α, and CCL-2. The level is compared to a reference level previously determined in the patient prior to therapy or at a previous time point during therapy. A change in the level compared to the reference is identified as indicating responsiveness to the therapy.
Another aspect of the invention is a kit for diagnosing Irritable Bowel Syndrome (IBS). In a divided or undivided container are five or more antibodies which specifically bind to a distinct cytokine selected from the group consisting of IL-5, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17, and CCL-2.
Still another aspect of the invention is a kit for distinguishing between Irritable Bowel Syndrome (IBS) and Irritable Bowel Disease (IBD). In a divided or undivided container are three or more antibodies which specifically bind to a distinct cytokine selected from the group consisting of IL-6, IL-10, IL-12, TNF-α, and CCL-2.
Yet another aspect of the invention is a kit for monitoring response to a therapy for Irritable Bowel Syndrome (IBS). In a divided or undivided container are three or more antibodies which specifically bind to a distinct cytokine selected from the group consisting of IL-1β, IL-6, IL-12, TNF-α, and CCL-2.
A further aspect of the invention is a device to aid in diagnosing Irritable Bowel Syndrome (IBS). It comprises five or more antibodies which specifically bind to a distinct cytokine selected from the group consisting of IL-5, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17, and CCL-2, and a means of detection of antibody binding to a sample component.
Still another aspect of the invention is a device to aid in distinguishing between Irritable Bowel Syndrome (IBS) and Irritable Bowel Disease (IBD). It comprises three or more antibodies which specifically bind to a distinct cytokine selected from the group consisting of IL-6, IL-10, IL-12, TNF-α, and CCL-2; and a means of detection of antibody binding to a sample component.
Another aspect of the invention is a device to aid in monitoring response to a therapy for Irritable Bowel Syndrome (IBS). The device comprises three or more antibodies which specifically bind to a distinct cytokine selected from the group consisting of IL-1β, IL-6, IL-12, TNF-α, and CCL-2; and a means of detection of antibody binding to a sample component.
These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with methods, kits, and devices for diagnosing or monitoring or predicting that an individual has or will develop IBS.
The inventors have developed methods of diagnosing, predicting, monitoring an individual who has or who will develop IBS. The methods use selected sets of cytokines which are distinctive for comparisons with normal unaffected individuals and with individuals who have similar but different disease pathology. Sets of cytokines can also be followed over time to see changes in the disease state of an individual with IBS.
Patient samples which may be analyzed in the methods include peripheral blood, serum, plasma, tissue samples, or other body fluid sample, such as stool, CSF, tears, saliva, and lymph. The patient may have mild, intermediate, or severe disease symptoms. Predefined levels for normal unaffected individuals, for affected IBS individuals, and for affected IBD individuals may include a median level or a range of levels of the cytokine or cytokine set found in such samples. The samples may be from a healthy subject, a diseased patient, or a patient having associated symptoms of IBS, and/or extra-intestinal involvement. Typically samples will be collected in a clinic and transferred to a laboratory for analysis. Once results are obtained, they may be transmitted back to the clinic or to the individual patient. Such transmission may be by any means, including electronic, oral, telephonic, or written. Results may be transmitted as raw data, as processed data, and/or as a conclusion.
The terms “indicates” or “correlates” in reference to a parameter, e.g., a modulated proportion, level, or cellular localization in a sample from a patient, may mean that the patient has IBS. The term “comparing” refers to making an assessment of how the proportion, level or cellular localization of one or more cytokines in a sample from a patient relates to the proportion, level or cellular localization of the corresponding one or more cytokines in a standard or control sample. The parameter may comprise the presence, absence and/or particular amounts of one or more cytokines. In other embodiments a parameter may comprise a weight in a multivariate algorithm (e.g. BOOSTED models, C&RT, Random Forests, Penalized regression models). The term “pattern” may mean a multivariate algorithm. A particular set or pattern of one or more cytokines (including the presence, absence, and/or particular amounts) may indicate that a patient has IBS (or correlates to a patient having IBS).
Different sets of cytokines have been identified which appear to be optimum for a particular type of determination. For example, where the disease state to be determined is IBS relative to healthy unaffected individuals, the cytokine set comprises one or more cytokines selected from the group consisting of interleukin IL-5, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17, and CCL-2 (MCP-1). Any whole number of these cytokines can be assessed, from 1 to 8, inclusive. Typically a single index is derived from the cytokine levels of the set. Where the disease states to be distinguished are IBS and IBD, the cytokines of the set can be selected from the group consisting of interleukin IL-6, IL-10, IL-12, TNF-α, and CCL-2(MCP-1). Any whole number of these cytokines from 1 to 5, inclusive, can be tested and the data combined into a single index. Where the disease state is IBS, and therapeutic monitoring is desired, the cytokine set can be selected from the group consisting of interleukin IL-1β, IL-6, IL-12, TNF-α, and CCL-2 (MCP-1). Any whole number of these cytokines from 1 to 5 may be used, inclusive, and the data combined into a single index. Various algorithms may be used to provide the single index. Optionally, the single index reflecting levels of cytokine expression may be combined with other clinical assessments, for example, pain, constipation, or diarrhea.
Antibodies can conveniently, but not exclusively, be used in characterizing the cytokine contents of various patient samples. Exemplary techniques which can be used include Enzyme Linked Immunosorbent Assay (ELISA) and its derivatives. Additionally, the antibody can be used in immunoblot or Western blot analysis (WB). The antibodies of the present invention may also be used in conjunction with fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks, such as blocks prepared from a tumor biopsy, prepared for study by immunohistochemistry (IHC). Another form of immunoassay involves protein array technology, which allows high-throughput screening. Typically these employ an array of antibodies for specifically capturing proteins. Non-antibody based assays which can be used for cytokines are bioassays which test for a cytokine's effect on particular cells. The cytokine biomarkers may optionally be detected by mass spectrometry or alternatively by means of an electrochemical luminescent assay. Other detection means that can be employed include optical methods, electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods.
Methods for analyzing levels of RNA typically are performed by specific hybridization, either to the RNA itself or to its reverse transcription product, cDNA. Specific probes may be used which are complementary to the RNA or to one or both strands of the cDNA. The sequences of the cytokine encoding genes are known in the art and can be used to design probes. Probes can be used in liquid or solid phase hybridization, such as on a nucleic acid array or on microparticles. Such methods are well known in the art.
Kits which can be formulated specifically to practice the methods of the invention may contain separate antibodies or nucleic acid probes or arrays or other aggregate or composite reagents. The kits may include other reagents for running the assays, including for example, secondary antibodies, labels, chromogenic reagents, chromophores, solid phases for binding or for separating binding products or cytokines prior to binding, including gels, chromatography matrices, buffers, enzymes, etc. Instructions for running assays including standard values (medians, ranges, etc.) for normal or diseased patients may also be provided as part of the kit. Because the kits are specifically formulated for the purposes of using the sets of cytokines disclosed here, they will not contain probes for the entire proteome or the entire transcriptome. Rather they will contain antibodies or probes for less than 100, less than 75, less than 50, less than 40, less than 30, less than 20, less than 15, or less than 10, less than 8 or less than 5 cytokines or cytokine encoding sequences.
Devices can be used for running the analyses as disclosed for the selected sets of cytokines. The devices may contain an array of antibodies or nucleic acid probes. The devices may contain detection means for identifying and quantitating binding of an analyte to the antibody or probe. The detection means may be any known in the art suitable for detecting fluorescence, radioactivity, color, heat, etc. Optionally, but desirably, the device will contain software for combining the results of levels of the determined cytokines or cytokine expression. Optionally, but desirably, the device will contain software for comparing the results between test samples and control samples. Optionally, the device will contain an output means for providing results, such as electronically, on paper, audibly, etc.
The power of a diagnostic test to correctly predict status is measured as the sensitivity or specificity of the assay or the area under a receiver operated characteristic (“ROC”) curve. The cytokine panels of the present invention may show a statistical difference in different IBS statuses of at least p<0.05, p<10-2, p<10-3, p<10-4 or p<10-5. Diagnostic tests that use these cytokines may show an ROC of at least 0.6, at least 0.7, at least 0.8, at least 0.9. The values measured for markers of a cytokine panel may be mathematically combined and the combined value correlated to the underlying diagnostic question. Advanced multivariate analyses including those of cluster analysis, factor analysis, discriminant function analysis (DFA), and multidimensional scaling were used to provide detailed characterization of cytokine-based IBS disease profiles and can be used to make diagnoses and evaluations. Cytokine measurements may be compared with relevant diagnostic amounts, cut-offs, ranges, or multivariate model scores that distinguish a positive IBS status from a negative IBS status.
Complementary multivariate analytical methods provide a vivid picture of the biological significance of the immune profile network. A DFA is distinct from the above analyses in that it is a class distinction modeling method that identifies sets of variables that best discriminate predefined disease and treatment groups. Multidimensional scaling provides a means of identifying correlational configurations of statistically significant cytokines, and allows for a visual representation of the pattern of proximities within the groups studied. These methods and analytical tools were utilized to identify novel diagnostic discriminatory cytokine biomarkers that can be used to distinguish sufficiently one IBS disease subtype from each other and controls. Furthermore, these tools were utilized to develop diagnostic, prognostic, disease activity-based, predictive, and therapeutic response panel of markers in patients with IBS disease subtypes.
Multidimensional scaling is an iterative process to detect meaningful underlying dimensions to explain observed similarities or dissimilarities between the groups studied [Borg I, Groenen P J F. Modern Multidimensional Scaling: Theory and Applications (Springer Series in Statistics). Springer; 2005]. This analysis uses correlational matrices to construct configurations of the data in a lower dimensional matrix, such that the relative distances between the groups are similar to those in the higher dimensional matrix. The degree of correspondence between the distances and the matrix input by the user is measured (inversely) by a stress function defined by Phi=Σ[dij−f(δij)]2, where dij stands for the euclidean distance, and δij stands for the observed distance. The proximities and distances are then represented on a two-dimensional Shepard diagram scatterplot which facilitates visualization and the interpretation of patterns. All statistical analyses for MDS were performed with R software [Ihaka R, Gentleman R. R: A Language for Data Analysis and Graphics. Journal of Computational and Graphical Statistics. 1996; 5:299-314].
As depicted in
Discriminant Function Analysis (DFA) is a multivariate class distinction algorithm that allows one to construct a mathematical model of discrimination built in a stepwise manner. This analysis was used here to identify the cytokines that best discriminated between patients with IBS and controls, and was modeled as previously described [(Alex P, Szodoray P, Knowlton N et al. Multiplex serum cytokine monitoring as a prognostic tool in rheumatoid arthritis. Clin Exp Rheumatol. 2007; 25:584-592.), (Szodoray P, Alex P, Jonsson M V et al. Distinct profiles of Sjogren's syndrome patients with ectopic salivary gland germinal centers revealed by serum cytokines and BAFF. Clin Immunol. 2005; 117:168-176.), (Jarvis J N, Dozmorov I, Jiang K et al. Novel approaches to gene expression analysis of active polyarticular juvenile rheumatoid arthritis. Arthritis Res Ther. 2004; 6:R15-R32.)]. Specifically, at each step, all variables are reviewed to determine which will maximally discriminate among groups. These variables are then included in a discriminative function, denoted a root, which is an equation consisting of a linear combination of cytokine changes used for the prediction of group membership. Variables will continue to be included in the model, as long as the respective F values for those variables are larger than the standard threshold (established by the analytical package Statistica, StatSoft, Tulsa, Okla., USA). The discriminant potential of the final equation from the forward stepwise DFA can then be observed in a simple multidimensional plot of the values of the roots obtained for each group. This multivariate approach identifies groups of analytes, the changes of which in levels can delineate profiles and create diagnostic patterns.
The utility of this analysis is that it identifies groups of analytes, the changes of levels in which can delineate profiles and create diagnostic patterns in IBS. Results of DFA can be visualized on a multidimensional plot, with class discrimination power represented by distance between both subtypes of IBS. All disease states were readily distinguished from controls (
To develop an index of cytokine levels using the results of these multivariate analyses that could be readily interpreted in a clinical context for the medical community, a scoring system was created, denoted the IBS Activity Index (IBSAI). These values represent the levels deduced from an algorithm containing an aggregate of relevant cytokine measurements where the root values from the discriminant functional analysis were normalized such that the maximal value of controls was 0. The normal IBSAI range was calculated in a standard manner, e.g., normal range=the 25-75% interquartile range of unaffected control values.
The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.
We assayed for inflammatory mediators in patients with IBS, and evaluated their role of these mediators in the immunopathogenesis of the disease. The results provide a vivid understanding of the mechanisms and mediators involved in the GI tract in IBS, and generate profiles that can enable effective diagnosis of IBS. Cytokine profiles that were identified at the systemic level were also validated locally in colonic tissues. We used multivariate approaches that further identified patterns that can uniquely distinguish IBS from IBD, which is a common differential diagnosis for IBS. We further evaluated the potential of these cytokines in therapeutic prognosis for IBS, and were able to identify profiles that characterize both response and non-response for patients with IBS. While some individual cytokines may have previously been associated with the syndrome, we found multi-cytokine patterns which can be used to diagnose, correlate with disease activity, and facilitate prognosis in IBS.
We performed multiplex serum cytokine profiling from serum of IBS patients and controls. The cohort also included unaffected age and sex matched unaffected controls. The following 24 cytokines were assessed: IL-1ra, IL-1α, IL-β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, IFN-γ, TNF-α, G-CSF, GM-CSF, IL-8, MIP-1α, MIP-1β, MCP-1 (also known as CCL-2), EGF, VEGF, FGF-basic, IP-10, and Eotaxin. (Invitrogen)
As shown in
To evaluate the potential of the cytokine profiles to discriminate IBS from that of unaffected controls, a multivariate analysis called discriminant functional analysis was used for selection of the set of analytes that maximally discriminate among IBS and controls built in a step-wise manner. This was then included in a discriminative function, denoted a root, which is an equation consisting of a linear combination of changes in analytes used for the prediction of group membership, and as shown in
To investigate whether treatment with personalized approaches may play a role in the effective mediation of the identified immunomodulatory profiles, IBS patients in a clinical trial treatment with clinical placebo effects (placebo acupuncture alone, or placebo acupuncture with an augmented patient-practitioner relationship) were followed through three clinical visits. Responders and non-responders were identified based on clinical indices including global improvement scale (range 1-7), adequate relief of symptoms, symptom severity score, and quality of life. When the discriminatory cytokine profiles were correlated with clinical scores, they were able to significantly differentiate response from non-response as shown in
The disclosure of each reference cited is expressly incorporated herein.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US2012/021172 | 1/13/2012 | WO | 00 | 1/17/2014 |
Number | Date | Country | |
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61432358 | Jan 2011 | US |