Sarcoidosis detection and treatments

Abstract

Provided herein are methods for the detection and treatment of sarcoidosis, and more particularly, to method to determine whether a subject with have a low or high sarcoidosis disease burden by applying an algorithm to the measured protein expression, wherein the algorithm compares the measured protein expression levels to a reference level of a subject without sarcoidosis, wherein the algorithm is selected from a machine learning algorithm, a clustering algorithm, a support vector machine, or combinations thereof. A treatment is selected based on the comparison predicting if the subject has sarcoidosis that will resolve without treatment (low sarcoidosis disease burden), of if the sarcoidosis will lead to severe sarcoidosis (high sarcoidosis disease burden).

Description

SARCOIDOSIS DETECTION AND TREATMENTS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a non-provisional patent application of and claims priority to U.S. provisional patent application serial number 63 / 550,456 filed on February 6, 2024, the contents of which are incorporated by reference in its entirety. TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates in general to the field of sarcoidosis, and more particularly, to detecting, measuring, and determining the severity of sarcoidosis and treatments thereof. STATEMENT OF FEDERALLY FUNDED RESEARCH

[0003] This invention was made with government support under R01HL113326 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0004] Without limiting the scope of the invention, its background is described in connection with sarcoidosis.

[0005] Becker et al., in a manuscript entitled “Serum cytokines and their predictive value in pulmonary involvement of systemic sclerosis” [Sarcoidosis Vasc Diffuse Lung Dis. 2019;36(4):274-284. doi: 10.36141 / svdld.v36i4.7612. Epub 2019 May 1. PMID: 32476963; PMCID: PMC7247092], measured serum cytokines (IL-6, IL-7, IL-8, IL-10, CCL2, CCL4, TGF- β, TNF-α) in 125 SSc patients, who were recruited and observed in an outpatient clinic. Of these, 60 had pulmonary involvement, classified as either interstitial lung disease (ILD, 43 patients), pulmonary arterial hypertension (PAH, 7 patients) or pulmonary hypertension and ILD (PH-ILD, 10 patients). The association of serum cytokines with clinical features was analyzed and a correlation with BAL cytokines measured in a subset of SSc patients with ILD. These authors found that IL-6 and IL-7 were predictive for death in a Cox regression analysis in all SSc patients as well as in all patients with pulmonary involvement; IL-6 was predictive for mortality in SSc- ILD patients.

[0006] Mortaz et al., in a manuscript entitled “Association of serum TNF-α, IL-8 and free light chain with HLA-DR B alleles expression in pulmonary and extra-pulmonary sarcoidosis.” [J Inflamm (Lond). 2015 Mar 19;12:21. doi: 10.1186 / s12950-015-0066-3. PMID: 25866481; PMCID: PMC4393873], found that in 16 patients with HLA DRB alleles only *7 and *12 were different in sarcoidosis patients. The levels of TNF-α and IL-8 in pulmonary sarcoidosis patients were higher than in EPS (P < 0.05) whereas the levels of FLC subunits in EPS were higher than in pulmonary sarcoidosis.

[0007] U.S. Pat. No.11,524,950, issued to Schafer, et al., entitled, “Treatment of immune-related and inflammatory diseases”, is said to teach methods of using compounds and compositions for modulating leukocytic activity, including activity of B cells and / or T cells monocytes, macrophages, and other lymphoid or myeloid-derived cell types, in immune-related diseases or inflammatory diseases. The patent is also said to teach pharmaceutical compositions and dosing regimens for use in such methods of treatment.

[0008] Despite these advances, a need remains for novel methods, biomarkers, algorithms, and methods of treating sarcoidosis. SUMMARY OF THE INVENTION

[0009] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of treating a subject suspected of having sarcoidosis, the method comprising: obtaining or having obtained a biological sample from the subject; measuring protein expression levels of one or more cytokine markers selected from: IFN^, BAFF, IL-13, IL-7, IP10, VEGF, IL-17, IL- 10, IL-1^, IL-4, G-CSF, RANTES, IL-6, FGF-basic, MIP1a, TGF-^2, TNF alpha, IL1RA, IL-8, or IL-15 in the biological sample; applying an algorithm to the measured protein expression, wherein the algorithm compares the measured protein expression levels to a reference level of a subject without sarcoidosis, wherein the algorithm is selected from a machine learning algorithm, a clustering algorithm, a support vector machine, or combinations thereof; and based on the comparison predicting if the subject has sarcoidosis that will resolve without treatment (low sarcoidosis disease burden), or if the sarcoidosis will lead to severe sarcoidosis (high sarcoidosis disease burden); if the subject has sarcoidosis that will resolve without treatment, then no treatment is provided; or if the subject has sarcoidosis that will lead to severe sarcoidosis then providing the subject with at least one of: a disease-modifying anti-rheumatic drugs (DMARDs), biologic DMARDs, non-tumor necrosis factor inhibitors (TNFi) biologics, or targeted syntheticDMARDs in an amount sufficient to reduce one or more symptoms of sarcoidosis. In one aspect, the support vector machine algorithm was trained with a radial basis kernel. In another aspect, the method further comprises determining if the subject is of African American ancestry and then further measuring MCP1 protein levels compared to the reference levels that are indicative of sarcoidosis in the subject. In another aspect, the method further comprises determining if the subject is of European American ancestry and then further measuring a Type 2 (IL-4 and IL-13), a Type 3 (IL-17) immune response, or both, compared to the reference levels are indicative for the presence of sarcoidosis in the subject. In another aspect, the biological sample comprises peripheral blood mononuclear cells (PBMCs) that are further divided into monocytes, T cells, and B cells, and measuring release of one or more cytokines before and after activation of the monocytes, T cells, and B cells. In another aspect, the biological sample is blood, plasma, a body fluid, or a biopsy. In another aspect, the protein expression is measured using an assay selected from at least one of: multiplex bead-based assay, capillary Western Blot, ELISA, flow cytometry, precipitin, fluorimetry, microscopy, immunofluorescence, radioimmunoassay, immunoenzymatic assay, fluorescence-activated cell sorting (FACS), differential display, representational difference analysis, microarray, Western blotting, immunohistochemical staining, immunocytochemical staining, dot blots, or surface plasmon resonance detection. In another aspect, the detection of sarcoidosis is based on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 biomarkers. In another aspect, the order of importance of the biomarkers in the sarcoidosis disease burden score is: IFN^, BAFF, IL-13, IL-7, IP10, VEGF, IL-17, IL-10, IL-1^, IL-4, G-CSF, RANTES, IL-6, FGF-basic, MIP1a, TGF-^2, TNF alpha, IL1RA, IL-8, and IL-15. In another aspect, the sarcoidosis disease burden score is higher when IFN^, IL-1^, and VEGF are elevated. In another aspect, the treatment of sarcoidosis is selected from at least one of: methotrexate, azathioprine, hydroxychloroquine, leflunomide, sulfasalazine, mycophenolate, cyclosporine, minocycline, tacrolimus, etanercept, adalimumab, infliximab, golimumab, certolizumab, abatacept, rituximab, secukinumab, ustekinumab, omalizumab, anakinra, tocilizumab, sarilumab; tofacitinib, baricitinib, or apremilast.

[0010] As embodied and broadly described herein, an aspect of the present disclosure relates to a method for predicting if a subject with sarcoidosis will have a low or a high disease burden and treating the subject, the method comprising: calculating a sarcoidosis disease burden score by scoring three measures of clinical disease selected from: (a) disease activity selected from resolved (0), mild but present (1), or persistent activity (2); (b) treatment requirements selectedfrom never (0), none for over a year (1), or currently under treatment (2); and (c) symptoms selected from asymptomatic (0), mildly symptomatic (1), moderate state symptoms (2), or worsening symptoms (3), determining if the subject has a low or a high sarcoidosis disease burden score based on the sum of (a)+(b)+(c), wherein a low disease burden score (^5) is predictive of mild sarcoidosis and a high disease burden score (^6) is predictive of severe sarcoidosis; and if the subject with a low sarcoidosis disease burden score, then no treatment or over-the-counter treatment is provided; or if the subject has a high sarcoidosis disease burden score, then the patient is treated with at least one of: disease-modifying anti-rheumatic drugs (DMARDs), biologic DMARDs, non-tumor necrosis factor inhibitors (TNFi) biologics, or targeted synthetic DMARDs in an amount sufficient to reduce one or more symptoms of sarcoidosis. In one aspect, the biological sample is blood, plasma, a body fluid, or a biopsy. In another aspect, the treatment of sarcoidosis is selected from at least one of: methotrexate, azathioprine, hydroxychloroquine, leflunomide, sulfasalazine, mycophenolate, cyclosporine, minocycline, and tacrolimus; etanercept, adalimumab, infliximab, golimumab, certolizumab, abatacept, rituximab, secukinumab, ustekinumab, omalizumab, anakinra, tocilizumab, sarilumab; tofacitinib, baricitinib, or apremilast. In another aspect, the symptoms in the lung are selected from mediastinal lymph node enlargement, pulmonary nodules, interstitial lung disease, and pulmonary fibrosis, cough, shortness of breath, and wheezing. In another aspect, the symptoms in the skin are selected from papules, nodules, plaques, lupus pernio, and infiltration of scars or tattoos. In another aspect, the symptoms in the eyes are selected from anterior, intermediate, or posterior uveitis, conjunctival nodules, lacrimal gland enlargement, painful and / or red eyes and vision loss. In another aspect, the symptoms in the liver are selected from abnormal liver function tests and hepatomegaly, abdominal pain, and jaundice. In another aspect, the symptoms in the lymph nodes are selected from enlargement of intrathoracic, abdominal, and peripheral lymph nodes. In another aspect, the symptoms in the spleen are selected from spleen enlargement, or abdominal pain. In another aspect, the symptoms in the heart are selected from atrioventricular or bundle branch block, tachyarrhythmias, congestive heart failure, shortness of breath, syncope, and sudden death. In another aspect, the symptoms in the nervous system are selected from facial nerve palsy, optic neuritis, trigeminal neuralgia, meningitis, hydrocephalus, spinal cord disease, polyneuropathy, and small-fiber neuropathy, facial palsy, vertigo, numbness, seizures, headache, hearing loss, visual and hearing loss, and weakness. In another aspect, the symptoms in the otherorgans are selected from kidney, salivary glands, ear-nose-throat, bone, bone marrow, joints, skeletal muscle, gastrointestinal tract, and genitourinary tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

[0012] FIG. 1 is a table that shows the cytokine association in all cases / controls. EA cases / controls, and high / low disease burden.

[0013] FIGS. 2A and 2B show the results from using a machine learning algorithm (Support Vector Machines, SVM) to construct predictive models for case status (FIG.2A) and high disease burden (FIG.2B).

[0014] FIG.3 shows the baseline (pre-stimulation) levels of both VEGF and IL-8 were reflective of circulating plasma cytokine levels (graphs on the left). The high, mid, and low plasma cytokine level groups secreted IL-6, VEGF, and IL-8 after stimulation confirmed that the circulating plasma cytokines are reflective of immune cell cytokine production (graphs on the right). DETAILED DESCRIPTION OF THE INVENTION

[0015] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0016] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

[0017] As used herein, the term “biomarker” or “biomarkers” refer to one or more characteristics that are objectively measured and evaluated as indicators of a normal or abnormal biological process, pathogenic (disease) processes, or pharmacologic responses to therapeutic interventions.As used in the context of sarcoidosis patients, the biomarkers are differentially expressed in patients with sarcoidosis.

[0018] As used herein, the terms “detectable”, “detectable biomarkers”, or “detectable labels” are used interchangeably to refer to directly or indirectly detecting a compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., a protein, element, or other molecule, such as an antibody or enzyme to generate a “labeled” composition. Detectable compounds and / or elements can be detected due to their specific functional properties and / or chemical characteristics, the use of which allows the agent to which they are attached or attachable to be detected, and / or further quantified if desired, such as, e.g., an enzyme, radioisotope, electron dense particles, magnetic particles or chromophore. There are many types of detectable labels, including fluorescent labels, which are easily handled, inexpensive, and nontoxic. The detectable portion can be attached to, e.g., an antibody that is specific for human antibodies, such that it forms a sandwich with the antigens, e.g., a sandwich ELISA or other secondary binding of agents to one or more detectable labels. The proteins can be measured using an assay selected from at least one of: multiplex bead-based assay, capillary Western Blot, ELISA, flow cytometry, precipitin, fluorimetry, microscopy, immunofluorescence, radioimmunoassay, immunoenzymatic assay, fluorescence-activated cell sorting (FACS), differential display, representational difference analysis, microarray, Western blotting, immunohistochemical staining, immunocytochemical staining, dot blots, or surface plasmon resonance detection.

[0019] As used herein, the terms “low sarcoidosis disease burden” and “high sarcoidosis disease burden” are used to characterize disease severity in patients with sarcoidosis (sarcoidosis symptoms, sarcoidosis disease activity, or treatment for sarcoidosis). What constitutes a “low sarcoidosis disease burden” or a “high sarcoidosis disease burden” is defined or determined as follows: (1) identify that the subject has sarcoidosis, (2) calculate a sarcoidosis disease burden score by scoring three measures of clinical disease selected from: (a) disease activity selected from resolved (0 points), mild but present (1 point), or persistent activity (2 points); (b) treatment requirements selected from never (0 points), none for over a year (1 point), or currently under treatment (2 points); and (c) symptoms selected from asymptomatic (0 points), mildly symptomatic (1 point), moderate state symptoms (2 points), or worsening symptoms (3 points), and finally, (3) calculate if the subject has a low or a high sarcoidosis disease burden score based on the sum of (a)+(b)+(c). A low disease burden score (^5) is predictive of mild sarcoidosis. A high disease burden score (^6) is predictive of severe or persistent sarcoidosis. Therefore, a lowdisease burden score (^5) is predictive of mild sarcoidosis and is indicative that the sarcoidosis will resolve without treatment, in which case no treatment is provided and / or minimal over-the- counter remedies are provided, those being a conservative treatment regime. However, if a high disease burden score (^6) is predictive of severe or persistent sarcoidosis, then the subject is provided with at least one of: a disease-modifying anti-rheumatic drugs (DMARDs), biologic DMARDs, non-tumor necrosis factor inhibitors (TNFi) biologics, or targeted synthetic DMARDs in an amount sufficient to reduce one or more symptoms of sarcoidosis.

[0020] As used herein, the term “disease burden” refers to an individual disease burden score reflective of the sarcoidosis impact on multiple aspects of an individual patient’s life. A “disease burden” score combines a snapshot of objective disease manifestations, patient-reported symptoms, and therapeutic requirements at a specific time point. Thus, a sarcoidosis patient with persistent or worsening symptoms, in need of disease-modifying treatment, and objective evidence of sarcoidosis is defined as having a high disease burden. A sarcoidosis patient that does not have persistent or worsening symptoms, does not need disease-modifying treatment, and has little to no objective evidence of sarcoidosis is defined as having a low disease burden score. The sarcoidosis disease burden score is calculated as shown in Table 1.

[0021] As used herein, the term “treating” refers to curing as well as ameliorating at least one symptom of sarcoidosis. Treatments for sarcoidosis may include a steroid, a disease-modifying antirheumatic drug (DMARD), immunosuppressants, or combinations thereof. For example, the present invention generates a disease burden score that distinguishes between levels of disease burden in patients with sarcoidosis. Table 1 below compares the definition of high and low of the present invention, and the historic cohort. As defined herein, based on clinical features in Table 1, a “low” score is equal or less than 5, while a “high” score is 6 or greater.

[0022] Based on the high or low scores, a patient with a high disease burden score is subject to the management of the disease or treatment with disease-modifying anti-rheumatic drugs (cDMARDs) including: methotrexate, azathioprine, hydroxychloroquine, leflunomide, sulfasalazine, mycophenolate, cyclosporine, minocycline, and tacrolimus; biologic DMARDs including tumor necrosis factor inhibitors (TNFi) (etanercept, adalimumab, infliximab, golimumab, certolizumab); non-TNFi biologics (abatacept, rituximab, secukinumab, ustekinumab, omalizumab, anakinra, tocilizumab, sarilumab); or targeted synthetic DMARDs (tsDMARDs) including: tofacitinib, baricitinib, and apremilast.

[0023] Signs and Symptoms of Organ Involvement in Sarcoidosis. Different symptoms are indicative of Sarcoidosis depending on the organ involved. The following list provides a list of symptoms for ach organ. 1. Lung – Mediastinal lymph node enlargement, pulmonary nodules, interstitial lung disease, and pulmonary fibrosis. Manifested by cough, shortness of breath, and wheezing. 2. Skin – Papules, nodules, plaques, lupus pernio, and infiltration of scars or tattoos. 3. Eyes – Anterior, intermediate, or posterior uveitis; conjunctival nodules; lacrimal gland enlargement. Manifested as painful and / or red eyes and vision loss. 4. Liver – Abnormal liver function tests and hepatomegaly. Often asymptomatic but may cause abdominal pain and jaundice. 5. Lymph nodes – Enlargement of intrathoracic, abdominal, and peripheral lymph nodes. Usually painless and identified through physical examination or imaging. 6. Spleen – Spleen enlargement, often asymptomatic or resulting in abdominal pain. 7. Heart – Atrioventricular or bundle branch block, tachyarrhythmias, and congestive heart failure. May be temporarily asymptomatic but can lead to shortness of breath, syncope, and sudden death. 8. Nervous system – Facial nerve palsy, optic neuritis, trigeminal neuralgia, meningitis, hydrocephalus, spinal cord disease, polyneuropathy, and small-fiber neuropathy. Manifested as facial palsy, vertigo, numbness, seizures, headache, hearing loss, visual and hearing loss, and weakness. 9. Other organs less frequently involved – Kidney, salivary glands, ear-nose-throat, bone, bone marrow, joints, skeletal muscle, gastrointestinal tract and genitourinary tract.

[0024] As used herein, the term “effective amount” refers to the amount of a compound or agent administered or delivered to the patient which is most likely to result in the desired treatment outcome. The amount is empirically determined by the patient's clinical parameters including, but not limited to the stage of disease, age, gender, histology, and likelihood for recurrence.

[0025] There is no gold standard biomarker for diagnosis or prognosis of sarcoidosis. By examining patients based on clinical / serologic subtype and ancestry, the present inventors identified biomarkers that can help in the diagnosis and prognosis of sarcoidosis, or provide more personalized treatment recommendations for sarcoidosis patients. Further, these same data gave insight into the mechanism of sarcoidosis initiation or progression.

[0026] The inventors measured, in circulation, 32 known immune cytokines or chemokines in 132 participants (~3:1 cases to controls). Performed scRNAseq on 210 participants, with approximately 72% of primarily European Ancestry and approximately 24% of primarily African Ancestry.

[0027] To show the usefulness of circulating cytokines in building models predictive of sarcoidosis susceptibility and disease burden, the inventors performed a study using plasma from ~100 patients and controls. FIG. 1 is a table that shows the cytokine association in all cases / controls. EA cases / controls, and high / low disease burden. It was found that almost half of the 32 cytokines tested were elevated in patients compared to controls (via logistic regression; p- value and Odds Ratio (OR) based on exponentiation of the regression coefficient shown in the table in FIG.1). The most significant effects were identified in the comparison of all cases to all controls, which had the largest sample size. However, several effects were found, even in the smaller stratified analyses (FIG.1). For example, 6 cytokines had larger OR were unique to the EA-only sample. MCP1, previously shown to be enriched in BAL of sarcoidosis patients and a regulatory mediator of inflammatory disease, was significant only in the AA sample (p<0.05). In addition, patients with high disease burden were more likely to exhibit elevated levels of IFN^, IL-1^, and VEGF than those with low disease burden – a finding not detected in cases compared to controls. Note that while the EA seem to show primarily a Type 2 (IL-4 and IL-13 ) and Type 3 (IL-17) immune response, all of these are less significant in the larger sample, suggesting that these effects are absent in AA, highlighting the need for expanded studies, and perhaps explaining the variability in even what are considered successful clinical trials. These results demonstratethat while a single factor may not have the sensitivity and specificity desired for clinically useful biomarkers, a panel of such biomarkers may exist for both diagnostic and prognostic usefulness.

[0028] To test this, the inventors used a machine learning method (Support Vector Machines, SVM) to construct predictive models for case status (FIG.2A) and high disease burden (FIG.1B). Before training the SVM model, the dataset was split into a training set and a test set with a 75 / 25 ratio. Implementing R package e1071, the SVM model will be trained using a radial basis kernel. Then the trained SVM model will be applied to the testing set to predict the class of each of its constituents. The accuracy, sensitivity, and specificity of the model will be calculated using the confusion Matrix function from the R package "Caret”. Susceptibility and disease burden models were trained and evaluated independently.

[0029] After performing a training and test set analysis, the inventors were able to predict case status with a sensitivity of 77% and specificity of 75%. Prediction of high disease burden had an even greater sensitivity (80%) but slightly less specificity (70%). The histograms show the contribution of each of the specific factors in each of the 2 models. The larger the weight, the more important the factor in defining the space that distinguishes one group from another.

[0030] PBMC Stimulation. 9 patients, 3 each of high, mid, and low levels (based on levels of the 12 cytokines were found statistically significant). PBMCs were thawed, seeded into 96-well culture plates (2x106cells / ml). The cells were stimulated using the toll-like receptor 4 agonist LPS (1ng / ml) for 24h, and then secreted cytokines were measured.

[0031] FIG.3 shows the baseline (pre-stimulation) levels of both VEGF and IL-8 were reflective of circulating plasma cytokine levels (graphs on the left). The high, mid, and low plasma cytokine level groups secreted IL-6, VEGF, and IL-8 after stimulation confirmed that the circulating plasma cytokines are reflective of immune cell cytokine production (graphs on the right). These responders all have high DB Score values and are currently treated with either steroids or DMARDs, suggesting detection even in the presence of immunosuppressive therapies.

[0032] Distinguishing Sarcoidosis Patients using Circulating Cytokines from Healthy Controls and Distinguishing Sarcoidosis Patients with high disease burden (DB).

[0033] The dataset contains samples from 85 Sarcoidosis patients and 36 healthy controls (36), 20 selected cytokines based on single variable association with either case / control status disease burden (DB) and ancestry. The inventors included participants who were genetically confirmedto be of European ancestry (EA), African ancestry (AA), or Central / Native American ancestry (CA).

[0034] Before training the support vector machine (SVM) model, the dataset was split into a training set and a test set with a 75 / 25 ratio, respectively (see Tables 1 and 2). With the help of the R package e1071, the SVM model was trained with the training set using a radial basis kernel. The trained SVM model was then used to predict the class in the testing set. The accuracy, sensitivity, and specificity of the model were calculated using the confusion Matrix function from the R package caret. Case / control and DB score _high / DB score_low were trained and evaluated independently (DBS – disease burden score).

[0035] As already described hereinabove, FIGS.2A and 2B show the contribution of each of the specific factors in each of the 2 models. The larger the weight, the more important a given factor is in defining the space that distinguishes one group from another.

[0036] The Tables below show the specifics of each model; sample size, sensitivity and specificity. The inventors were able to classify cases and controls with an accuracy of 0.7667, meaning that it correctly predicted the outcome for 76.67% of the testing set. The model had a sensitivity of 0.7692 (true positives) and a specificity of 0.75 (true negatives). In DBS _high / DBS _low, the accuracy, sensitivity, and specificity values are 0.75, 0.75, 0.75, respectively.

[0037] Table 2. Patients compared to Healthy Controls. num of features 21

[0038] Table 3. Patients with high DB Score compared to low DB Score. num of features 21 num of total samples 81

[0039] The inv ing these cytokinesand found differential expression in both the African American (AA) sample as well as the European American (EA) sample for the cases compared to controls and high compared to low DB Score values.

[0040] Table 4. Differential expression in both the African-American sample as well as the European-American sample. Case / Control

[0041] Spec8 and IL1B in monocytes and IFNg only in NK cells in African Americans.

[0042] Table 5. Differential expression in patients of high compared to low Disease Burden. High / Low DB ScoreCytokine Gene AA EA ALL

[0043] S , CCL5 in monocytes, B cells, T cells and Dendritic Cells and TNFSF13B only in monocytes in African Americans and Interferon gamma (IFNg) only in NK cells in African Americans.

[0044] A person of skill in the art would readily recognize that steps of various above-described methods can be performed by programmed computers. Herein, some embodiments are also intended to cover program storage devices, e.g., digital data storage media, which are machine or computer-readable and encode machine-executable or computer-executable programs of instructions, wherein said instructions perform some or all of the steps of said above-described methods. The program storage devices may be, e.g., digital memories, magnetic storage media such as magnetic disks and magnetic tapes, hard drives, or optically readable digital data storage media. The embodiments are also intended to cover computers programmed to perform said steps of the above-described methods.

[0045] The functions of the various elements shown in the figures, including any functional blocks labeled as "modules", may be provided through the use of dedicated hardware as well as hardware capable of executing software in association with the appropriate software. When provided by a processor, the functions may be provided by a single dedicated processor, by a single shared processor, or by a plurality of individual processors, some of which may be shared. Moreover, explicit use of the term "module" should not be construed to refer exclusively to hardware capable of executing software, and may implicitly include, without limitation, digital signal processor (DSP) hardware, network processor, application-specific integrated circuit (ASIC), field-programmable gate array (FPGA), read-only memory (ROM) for storing software, random access memory (RAM), and nonvolatile storage. Other hardware, conventional and / or custom, may also be included.

[0046] A risk score of the present invention may be calculated with an algorithm using well- known statistical analysis techniques. Non-limiting examples of statistical analysis techniques that may be used to calculate the risk score include cross-correlation, Principal Components Analysis (PCA), factor rotation, Logistic Regression (LogReg), Linear Discriminant Analysis (LDA), Eigengene Linear Discriminant Analysis (ELDA), Support Vector Machines (SVM), Random Forest (RF), Recursive Partitioning Tree (RPART), related decision tree classification techniques, Shrunken Centroids (SC), StepAIC, Kth-Nearest Neighbor, Boosting, Decision Trees, Neural Networks, Bayesian Networks, Support Vector Machines, and Hidden Markov Models, Linear Regression or classification algorithms, Nonlinear Regression or classification algorithms, analysis of variants (ANOVA), hierarchical analysis or clustering algorithms; hierarchical algorithms using decision trees; kernel based machine algorithms such as kernel partial least squares algorithms, kernel matching pursuit algorithms, kernel Fisher's discriminate analysis algorithms, or kernel principal components analysis algorithms. In preferred embodiments, the risk score may be calculated using a random forest algorithm using the concentrations of three or more sample analytes in the panel of biomarkers. In an exemplary embodiment, the risk score is calculated as described in the examples.

[0047] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0048] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0049] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0050] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0051] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of”. As used herein, the phrase “consisting essentially of” requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method / process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method / process steps or limitation(s)) only.

[0052] The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0053] As used herein, words of approximation such as, without limitation, “about”, "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art towarrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0054] Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the invention(s) set out in any claims that may issue from this disclosure. Specifically and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any invention(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the invention(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure, but should not be constrained by the headings set forth herein.

[0055] For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.

[0056] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.

[0057] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferredembodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

Claims

What is claimed is:

1. A method of treating a subject suspected of having sarcoidosis, the method comprising: obtaining or having obtained a biological sample from the subject; measuring protein expression levels of one or more cytokine markers selected from: IFN^, BAFF, IL-13, IL-7, IP10, VEGF, IL-17, IL-10, IL-1^, IL-4, G-CSF, RANTES, IL-6, FGF-basic, MIP1a, TGF-^2, TNF alpha, IL1RA, IL-8, or IL-15 in the biological sample; applying an algorithm to the measured protein expression, wherein the algorithm compares the measured protein expression levels to a reference level of a subject without sarcoidosis, wherein the algorithm is selected from a machine learning algorithm, a clustering algorithm, a support vector machine, or combinations thereof; and based on the comparison predicting if the subject has sarcoidosis that will resolve without treatment (low sarcoidosis disease burden) or if the sarcoidosis will lead to severe sarcoidosis (high sarcoidosis disease burden); if the subject has sarcoidosis that will resolve without treatment, then no treatment is provided; or if the subject has sarcoidosis that will lead to severe sarcoidosis then providing the subject with at least one of: a disease-modifying anti-rheumatic drugs (DMARDs), biologic DMARDs, non-tumor necrosis factor inhibitors (TNFi) biologics, or targeted synthetic DMARDs in an amount sufficient to reduce one or more symptoms of sarcoidosis.

2. The method of claim 1, wherein the support vector machine algorithm was trained with a radial basis kernel.

3. The method of claim 1, further comprising determining if the subject is of African American ancestry and then further measuring MCP1 protein levels compared to the reference levels that are indicative of sarcoidosis in the subject.

4. The method of claim 1, further comprising determining if the subject is of European American ancestry and then further measuring a Type 2 (IL-4 and IL-13), a Type 3 (IL-17) immune response, or both, compared to the reference levels are indicative for the presence of sarcoidosis in the subject.

5. The method of claim 1, wherein the biological sample comprises peripheral blood mononuclear cells (PBMCs) that are further divided into monocytes, T cells, and B cells, and measuring release of one or more cytokines before and after activation of the monocytes, T cells, and B cells.

6. The method of claim 1, wherein the biological sample is blood, plasma, a body fluid, or a biopsy.

7. The method of claim 1, wherein the protein expression is measured using an assay selected from at least one of: multiplex bead-based assay, capillary Western Blot, ELISA, flow cytometry, precipitin, fluorimetry, microscopy, immunofluorescence, radioimmunoassay, immunoenzymatic assay, fluorescence-activated cell sorting (FACS), differential display, representational difference analysis, microarray, Western blotting, immunohistochemical staining, immunocytochemical staining, dot blots, or surface plasmon resonance detection.

8. The method of claim 1, wherein the detection of sarcoidosis is based on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 biomarkers.

9. The method of claim 1, wherein the order of importance of the biomarkers in the sarcoidosis disease burden score is: IFN^, BAFF, IL-13, IL-7, IP10, VEGF, IL-17, IL-10, IL-1^, IL-4, G-CSF, RANTES, IL-6, FGF-basic, MIP1a, TGF-^2, TNF alpha, IL1RA, IL-8, and IL-15.

10. The method of claim Error! Reference source not found., wherein the sarcoidosis disease burden score is higher when IFN^, IL-1^, and VEGF are elevated.

11. The method of claim 1, wherein the treatment of sarcoidosis is selected from at least one of: methotrexate, azathioprine, hydroxychloroquine, leflunomide, sulfasalazine, mycophenolate, cyclosporine, minocycline, tacrolimus, etanercept, adalimumab, infliximab, golimumab, certolizumab, abatacept, rituximab, secukinumab, ustekinumab, omalizumab, anakinra, tocilizumab, sarilumab; tofacitinib, baricitinib, or apremilast.

12. A method for predicting if a subject with sarcoidosis will have a low or a high disease burden and treating the subject, the method comprising: calculating a sarcoidosis disease burden score by scoring three measures of clinical disease selected from: (a) disease activity selected from resolved (0), mild but present (1), or persistent activity (2); (b) treatment requirements selected from never (0), none for over a year (1), or currently under treatment (2); and (c) symptoms selected from asymptomatic (0), mildly symptomatic (1), moderate state symptoms (2), or worsening symptoms (3),determining if the subject has a low or a high sarcoidosis disease burden score based on the sum of (a)+(b)+(c), wherein a low disease burden score (^5) is predictive of mild sarcoidosis and a high disease burden score (^6) is predictive of severe sarcoidosis; and if the subject has a low sarcoidosis disease burden score, then no treatment or over-the- counter treatment is provided; or if the subject has a high sarcoidosis disease burden score, then the patient is treated with at least one of: disease-modifying anti-rheumatic drugs (DMARDs), biologic DMARDs, non- tumor necrosis factor inhibitors (TNFi) biologics, or targeted synthetic DMARDs in an amount sufficient to reduce one or more symptoms of sarcoidosis.

13. The method of claim 12, wherein the biological sample is blood, plasma, a body fluid, or a biopsy.

14. The method of claim 12, wherein the treatment of sarcoidosis is selected from at least one of: methotrexate, azathioprine, hydroxychloroquine, leflunomide, sulfasalazine, mycophenolate, cyclosporine, minocycline, and tacrolimus; etanercept, adalimumab, infliximab, golimumab, certolizumab, abatacept, rituximab, secukinumab, ustekinumab, omalizumab, anakinra, tocilizumab, sarilumab; tofacitinib, baricitinib, or apremilast.

15. The method of claim 12, wherein the symptoms in the lung are selected from mediastinal lymph node enlargement, pulmonary nodules, interstitial lung disease, and pulmonary fibrosis, cough, shortness of breath, and wheezing.

16. The method of claim 12, wherein the symptoms in the skin are selected from papules, nodules, plaques, lupus pernio, and infiltration of scars or tattoos.

17. The method of claim 14, wherein the symptoms in the eyes are selected from anterior, intermediate, or posterior uveitis, conjunctival nodules, lacrimal gland enlargement, painful and / or red eyes and vision loss.

18. The method of claim 12, wherein the symptoms in the liver are selected from abnormal liver function tests and hepatomegaly, abdominal pain, and jaundice.

19. The method of claim 12, wherein the symptoms in the lymph nodes are selected from enlargement of intrathoracic, abdominal, and peripheral lymph nodes.

20. The method of claim 12, wherein the symptoms in the spleen are selected from spleen enlargement, or abdominal pain.

21. The method of claim 12, wherein the symptoms in the heart are selected from atrioventricular or bundle branch block, tachyarrhythmias, congestive heart failure, shortness of breath, syncope, and sudden death.

22. The method of claim 12, wherein the symptoms in the nervous system are selected from facial nerve palsy, optic neuritis, trigeminal neuralgia, meningitis, hydrocephalus, spinal cord disease, polyneuropathy, and small-fiber neuropathy, facial palsy, vertigo, numbness, seizures, headache, hearing loss, visual and hearing loss, and weakness.

23. The method of claim 12, wherein the symptoms in the other organs are selected from kidney, salivary glands, ear-nose-throat, bone, bone marrow, joints, skeletal muscle, gastrointestinal tract, and genitourinary tract.