Field of the invention

[0001] The present invention relates to an in vitro diagnostic method for diagnosis of the risk of relapses in arthritis in a patient by combined testing of the patient sample for S100A12 and high sensitivity C- eactive Protein (hsCRP). The present invention relates to an in vitro diagnostic method for excluding the risk of developing arthritis relapses in a patient by combined testing of the patient sample for S100A12 and hsCRP.

[0002] Particularly, the present invention relates to an in vitro diagnostic method for diagnosis of the risk of relapses in juvenile idiopathic arthritis (JIA) in a patient by combined testing of the patient sample for S100A12 and high sensitivity C-Reactive Protein (hsCRP). The present invention relates to an in vitro diagnostic method for excluding the risk of developing JIA relapses in a patient by combined testing of the patient sample for S100A12 and hsCRP.

[0003] In the diagnostic methods of the invention the sensitivity and the negative predictive value of JIA relapses are increased.

[0004] Furthermore, the present invention provides methods for the treatment of arthritis and more in particular JIA relapses which comprise the inventive methods as an essential part of the treatments.

Background of the invention

[0005] Juvenile idiopathic arthritis (JIA) is a chronic disease characterized by relapsing joint inflammation in children. Patients can be subdivided into at least three major subgroups - oligoarticular, polyarticular, and systemic JIA - with a total of seven subcategories. Treatment strategies include nonsteroidal antiinflammatory drugs, corticosteroids, immunosuppressive agents, and biologicals. In most patients clinical remission is successfully induced, but others have a refractory course with severe destructive arthritis and complications related to treatment. Even if remission is induced, about half of the patients experience a relapse after drug withdrawal (off medication). Hence, patients in remission off medication have a so called "background risk" or "pretest risk" of 50% of experiencing JIA relapse after drug withdrawal.

[0006] According to an international consensus on preliminary criteria for inactive disease status in JIA, six continuous months of inactive disease while taking anti-inflammatory drugs defines "clinical remission on medication". The definition is based upon clinical examination and includes as standard laboratory parameters standard CRP and the erythrocyte sedimentation rate.

[0007] Presently, it is unclear, however, whether a therapy maintaining this status will improve the chances of a given individual to reach the final goal which is clinical remission off medication. "Clinical remission off medication" is currently defined as 12 months of inactive disease after all antiinflammatory drugs have been withdrawn.

[0008] Foell et al. (JAMA 2010;303:1266-73) demonstrated that the stability of remission off medication is not generally improved by a prolonged maintenance therapy with pharmaceutical treatment (e.g., with methotrexate) but rather depends on individual immunological predisposition of the patients.

[0009] Ideally, physicians would individually define a patient's remission status on the basis of the synovial inflammation. In clinical remission, a low degree of inflammatory activity may remain present even though this is not detectable by clinical or routine laboratory means. Relapses might evolve from subclinical disease activity, e.g. a status of clinical but not immunologic remission. In this situation, remission off medication will not be achievable, and therapy withdrawal will result in relapsing disease.

[0010] Commonly used laboratory parameters have no predictive value for the further course of the disease, Foell et al. (JAMA 2010;303:1266-73). For example, according to this study, CRP measured according to the conventional assays is not predictive for risk of relapses in J IA patients. CRP is a member of the class of acute phase reactants that respond to rising concentrations of circulating cytokines. For conventional CRP assays, test values are typically considered to be clinically significant at level above lOmg/L. In apparently healthy persons the blood CRP level are below 5mg/L ("Review Criteria for assessment of C-Reactive Protein, High Sensitivity C-Reactive Protein (hsCRP) and Cardiac C-Reactive Protein cCRP) Assays" issued by FDA on 22 September 2005).

[0011] Recent data (Foell et al. JAMA 2010;303:1266-73) revealed that biomarkers of innate immune activation may detect non-apparent disease activity and thereby inform about the risk for relapse. Molecular biomarkers of inflammation are the neutrophil activation marker S100A12, the phagocyte activation marker myeloid-related proteins 8 and 14 heterocomplex (MRP8/14; S100A8/A9; calprotectin).

[0012] The neutrophil activation marker S100A12 is expressed and secreted mainly by granulocytes and exhibits strong pro-inflammatory activity. Intracellular signaling via various pathways induces secretion of cytokines by monocytes and up-regulation of adhesion molecules on endothelial cells. Human CALGRANU LIN C, which is also called S100A12, EN-RAGE, CAAF1 and p6 pro- tein, is a small protein of 92 amino acids which belongs to the family of calcium-binding S100 proteins (Guignard et al., 1995, Biochem J 309 : 395-401 ; US 5,976, 832). Homologues of CALG RANU LI N C in other species are known from Bos taurus (US 5,976, 832), pig (Dell'Angelica, 1994, J Biol Chem 269: 28929-28936) and rabbit (partial sequence: Yang et al., 1996, J Biol Chem 271: 19802-19809). Like other S100 proteins, it is suggested to play a role in general inflammation, although the role in inflammation within the S 100 family is inconsistent in that some of them are inhibiting the function of inflammatory cells while others are activating. It was proposed that CALGRANU LIN C plays a proinflammatory role (Donato, 2001, Int J Biochem Cell Biol 33: 637-668; Donato, 1999, Biochim Biophys Acta, 81450: 191-231 ; Yang et al., 2001, J Leukoc Biol 69 : 986-994). S100 proteins accumulate at sites of inflammation, and high levels of S10OA8 (also referred to as myeloid-related protein 8, MRP8 or calgranulin A) and S100A9 (MRP14 or calgranulin B) are found in inflammatory diseases like rheumatoid arthritis, inflammatory bowel disease, and CF (Golden et al., 1996, Arch Dis Child 74 : 136-9; Frosch et al., 2000, Arthritis Rheum 43: 628-37; Roth et al., 2001, Lancet 357: 1041). Overexpression of murine S10OA8 was detected in a mouse model of CF (Thomas et al., 2000, JImmunol 164 : 3870-3877). CALGRAN ULI N C is expressed by granulocytes, whereas it's expression by monocytes re- mains controversial (Vogl et al., 1999, J Biol Chem 274: 25291-25296; Hofmann et al. 1999, Cell 97: 889-901; Yang et al., 2001, J Leukoc Biol 69 : 986-994; Robinson et al., 2000, Bio- chem Biophys Res Com7nun 275: 865-870). It is secreted by activated granulocytes (Boussac et al., 2000, Electrophoresis 21: 665-672). Extracellular functions include potent chemotactic activity comparable to other strongly chemotactic agents (Hofmann et al. 1999, Cell 97 : 889-901; Miranda et al., 2001, FEBS Lett 488: 85-90). CALGRANULIN C is a ligand for the receptor for advanced glycation end products (RAGE) expressed on macrophages, lymphocytes, and endothelium (Hofmann et al. 1999, Cell 97: 889-901). Intracellular signalling via protein kinases induces nuclear factor (NF) -kappa independent secretion of different cytokines (Yeh et al., 2001, Diabetes 50: 1495-1504; Lander et al., 1997, J Biol Chem 272: 17810-17814). Assays for determining S100A12 are disclosed for example in patent application WO03/069341.

[0013] MRP8/14 are calcium-binding proteins of the SlOO-family expressed by early recruited monocytes and granulocytes. The MRP8/14 complex is secreted by infiltrating phagocytes in synovial inflammation and acts as a pro-inflammatory ligand of Toll-like receptor 4. Serum concentrations of these proteins correlate with synovial inflammation in JIA, and in particular with the risk to develop a relapse after withdrawal of methotrexate.

[0014] High sensitivity C-Reactive Protein (hsCRP) assays detect C-Reactive Protein. HsCRP assays have a range of measurement that extends below the measurement range typical of conventional CRP assays. Presently indications for hsCRP assays are general and not associated with specific diseases or risk for diseases.

[0015] There is the need to identify molecular biomarkers or combinations thereof for determining the risk of arthritis relapse in a patient despite clinical remission. This need is of particular importance in JIA as it affects the young populations and may have consequence for the entire life of a patient

[0016] In particular, considering the fact that an arthritis patient is already faced with a back-ground (pre-test) high risk of experiencing arthritis relapse (e.g. the background risk of experiencing relapse is of 50% in JIA patient), there is the need to determine more precisely the individual risk of arthritis relapse off medication for each patient. More particularly, there is the need to determine more precisely the individual risk of relapse off medication for each patient. Therefore, there is the need of molecular biomarkers or combinations thereof that assess more precisely the risk of arthritis relapses in each single patient. There is the need of molecular biomarkers or combinations thereof that more precisely asses the risk of arthritis relapse of each single patient for allowing a correct on/off medication decision making process. There is therefore the need of diagnostic methods with an improved sensitivity and negative predictive values.

Summary of the invention

[0017] The present inventors have surprisingly found that low levels of CRP i.e. below the detection limit of a standard CRP assay (5mg/L) are of significance in determining the risk of arthritis relapse, preferably of JIA relapse in a patient. Amounts and/or concentrations of CRP below the detection limit of a standard CRP assay cannot be correctly detected with standard CRP assays. Hence, the use of an hsCRP assay to detect these amounts and/or concentrations is required for the assessment of the risk of arthritis relapse, preferably of JIA relapse and in the diagnostic exclusion of the risk of developing arthritis relapse, preferably JIA relapse.

[0018] Indeed, S100A12 and MRP8/14 are meaningful for prediction of the relapse risk in arthritis, preferably in JIA. The present inventors have shown that each of the single phagocyte activation markers S100A12 and MRP8/14 provides good diagnostic performance in the prediction of disease flares, if they are used separately. Both biomarkers are especially useful in predicting early flares within 3 months after therapy withdrawal.

[0019] The present inventors have surprisingly found that hsC P assays provide some additional information on later flares and that the predictive performance of the marker S100A12 is improved by adding the detection of the marker hsCRP. The best combined marker panel (as shown by the best Youden-lndex (Table 1) is "S100A12 and/or hsCRP".

[0020] More specifically, the present inventors have found that by combined testing for S100A12 and hsCRP both the sensitivity and the negative predictive value of risk of relapse can be increased with respect to the sensitivity and the negative predictive value of risk of relapse of the respective assays alone. Additionally, the improved values of the Youden-lndex of the combined tests with respect to the Youden-lndex of the single assays indicate an overall better performance of the combined tests.

[0021] JIA patients in remission off medication have a background or pre-test risk of JIA relapse. Nowadays the background (pre-test) risk is assessed to be about 50% (i.e., a patient has the 50% of probability of experiencing JIA relapse after drug withdrawal). In other words, a patient out of 2 is at risk of JIA relapse. Each patient however, has an individual baseline risk of JIA relapse which may be higher or lower than the background risk. The present inventors have found that with the methods of the invention the risk of JIA relapse associated with each individual patient can be more precisely determined. Moreover, since the risk of JIA relapse determined according to the present invention is based on biomarkers tested on each individual patient a better on/off medication decision making process for each single patient is achieved.

[0022] In the methods of the invention, both S100A12 and hsCRP are measured. When a measured value of S100A12 concentration (or amount) is below a pre-defined cut off value and the measured value of hsCRP concentration (or amount) is below a pre-defined cut off value, there is an indication of a decreased risk of experiencing arthritis relapses, preferably JIA relapse, or there is an indication of a low or of a lower risk of experiencing arthritis relapse, preferably JIA relapse. Preferably, the risk of experiencing arthritis relapse, preferably JIA relapse is equal or below 30% i.e. the patient has 30% or less of probability of experiencing arthritis relapse, preferably JIA relapse. More preferably, the risk of experiencing arthritis relapse, preferably JIA relapse is equal or less than 25%. i.e., the patient has 25% or less of probability of arthritis relapse, preferably JIA relapse off medication. Preferably, a patient having a low risk of relapses according to the invention is considered in stable remission.

[0023] When either or both of the measured value of S100A12 and of the measured value of hsCRP is/are equal or above the respective pre-defined cut off value there is an indication of an increased risk of arthritis relapse, preferably JIA relapse or there is an indication of a high or a higher risk of arthritis relapse, preferably JIA relapse in a patient. The risk of experiencing arthritis relapse, preferably JIA relapse can be equal or above 70%, more preferably equal or above 75% i.e. the patient has 70% or more or the patient has 75% or more of probability to experience arthritis relapse, preferably JIA relapse. Preferably, a patient having a high risk of arthritis relapse, preferably of JIA relapses according to the invention is considered in instable remission.

[0024] Hence, the methods of the invention, when performed on patients in clinical remission on medication, allow dividing clinical remission on medication arthritis patients, preferably JIA patients, in patients with a high risk of relapse and patients with a low risk of relapse, preferably of JIA relapse. More in particular, the methods of the invention allow dividing arthritis patients, preferably JIA patients in clinical remission on medication that potentially have a background (pre-test) risk of 50% (i.e., the same risk to experiencing or not experiencing relapse in an off medication status) in patients with an high risk of relapse and patients with a low risk of relapse, preferably of JIA relapse off medication.

[0025] Patients wherein S100A12 and hsCRP values are below the respective pre-defined cut off value are considered patients in stable remission while patient with S100A12 and/or hsCRP values equal or above the respective pre-defined cut off value are considered patients in instable remission.

[0026] Therefore, the methods of the invention allow the assessment of the risk associated with arthritis relapse, preferably JIA relapse for each patient. Preferably, relapse is relapse off medication. Therefore, the methods of the invention allow determining whether a patient is in stable or instable remission. Preferably, the methods of the invention allow determining whether a patient is in stable remission on medication or off medication and instable remission on medication or off medication. Therefore, the methods of the invention allow a correct "on medication"/"off medication" decision making process.

[0027] Hence, the present invention is directed to a method for the diagnosis of risk of arthritis relapse in a patient, comprising the following steps:

(a) testing of a sample from said patient with a reagent or reagents for the detection of S100A12 amount or concentration and with a reagents or reagents for the detection of hsCRP amount or concentration, and

(b) evaluating the values of the amounts or concentrations measured in step (a).

[0028] Preferably, in the method of the invention when a measured value of S100A12 is below a predefined cut off value and when a measured value of hsCRP is below a pre-defined cut off value there is an indication of a decreased risk of arthritis relapse or of a low or lower risk of arthritis relapse. Preferably, the risk of arthritis relapse is equal or below 30% i.e. the patient has 30% or less of probability of arthritis relapse off medication. More preferably, the risk of experiencing arthritis relapse is equal or less than 25%. i.e., the patient has 25% or less of probability of arthritis relapse off medication. Preferably, a patient having a low risk of arthritis relapses according to the invention is considered in stable remission.

[0029] Preferably, when a measured value of S100A12 is equal or above a pre-defined cut off value and/or when the measured value of hsCRP is equal or above a pre-defined cut off value there is an indication of an increased risk of arthritis relapse or of high or a higher risk of arthritis relapse. The risk of arthritis relapse can be above 70%, more preferably above 75% i.e., the patient has a probability of 70% or 75% or more of experiencing arthritis relapse off medication. Preferably, a patient having a high risk of arthritis relapses according to the invention is considered in instable remission. [0030] Hence, in an aspect the invention is directed to a method for the diagnostic risk of relapses in arthritis in a patient, comprising the following steps:

(a) testing of a sample from said patient with a reagent or reagents for the detection of S100A12 amount or concentration and with a hsC P reagents or reagents for the detection of hsCRP amount or concentration and evaluating the values of the amounts or concentrations measured in step

i) wherein when the measured value of S100A12 is below a pre-defined cut off value and the measured value of hsCRP is below a pre-defined cut off value, there is an indication of a low risk of arthritis relapse or ii) wherein when the measured value of S100A12 and/or the measured value of hsCRP is equal or above a pre-defined cut off value there is an indication of a high risk of arthritis relapse.

[0031] A low or lower risk of arthritis relapse is associated with a stable remission in a patient. It is also associated with an immunological remission of the patient. Preferably, the risk of arthritis relapse is equal or below 30% i.e. the patient has 30% or less of of probability of arthritis relapse off medication. More preferably, the risk of experiencing arthritis relapse is equal or less than 25%. i.e., the patient has 25% or less of probability of arthritis relapse off medication. Preferably, a patient having a low risk of arthritis relapses according to the invention is considered in stable remission.

[0032] A high or higher risk of arthritis relapse is associated with an instable remission in a patient. It is also associated with a non-immunological remission of the patient. Preferably, a high or higher risk of experiencing arthritis relapse is a risk equal or greater than 70%, preferably equal or greater than 75% of arthritis relapse, preferably of arthritis relapse off medication i.e., the patient has a probability equal or greater than 70% or 75% to experience arthritis relapse preferably off medication. Preferably, a patient having a high risk of arthritis relapses according to the invention is considered in instable remission.

[0033] In an aspect, the present invention is directed to a method for the diagnosis of risk of relapses in arthritis in a patient, comprising the following steps:

(a) testing of a sample from said patient with a reagent or reagents for the detection of S100A12 amount or concentration and with a hsCRP reagents or reagents for the detection of hsCRP amount or concentration and

(b) evaluating the values of the amounts or concentrations measured in step (a) wherein

(i) when the measured value of S100A12 is below a pre-defined cut off value and the measured value of hsCRP is below a pre-defined cut off value there is a risk of arthritis relapse lower than the risk of arthritis relapse of when a measured value of S100A12 and/or a measured value of hsC P is (are) equal or above a respective pre-defined cut off value or

(ii) when the measured value of S100A12 and/or the measured value of hsCRP is (are) equal or above a respective pre-defined cut off value, there is a risk of arthritis relapse greater than the risk of arthritis relapse of when

a measured value of S100A12 and a measured value of hsCRP are below a respective pre-defined cut off value.

[0034] The hazard ratio (HR) characteristic of the combined tests can be used to determine how high is the risk of arthritis relapses of one patient or of a group of patients (e.g., the patient or the group of patients with a S100A12 value and/or a hsCRP value above the respective cut off) with respect to a patient or a group of patients with both S100A12 value and hsCRP value below the respective cut off.

[0035] Hence, in an aspect in the methods of the invention, when the measured value of S100A12 is below a pre-defined cut off value and the measured value of hsCRP is below a pre-defined cut off value there is a risk of arthritis relapse about three times lower than the risk of arthritis relapse of when a measured value of S100A12 and/or a measured value of hsCRP is (are) equal or above a respective pre-defined cut off value. In an aspect of the invention, when the measured value of S100A12 and/or the measured value of hsCRP is (are) equal or above a respective pre-defined cut off value, there is a risk of arthritis relapse about three times greater than the risk of arthritis relapse of when the measured value of S100A12 is below a pre-defined cut off value and the measured value of hsCRP is below a pre-defined cut off value.

[0036] In an aspect, the present invention is directed to a method for the diagnostic exclusion of the risk of developing relapse in arthritis in a patient, comprising the following steps:

(a) testing of a sample from said patient with a reagent or reagents for the detection of S100A12 amount or concentration and with a reagent or reagent for the detection of hsCRP amount or concentration and

(b) evaluating the values of the amounts or concentrations measured in step (a).

[0037] Preferably, the measured value of S100A12 amount or concentration and the measured value of hsCRP amount or concentration both below the respective pre-defined cut off values are indicative of stable remission.

[0038] Preferably, the measured values of S100A12 and/or of hsCRP amount or concentration, one or both above or equal to the respective pre-defined cut off values are indicative of instable remission. [0039] Hence, in an aspect the present invention is directed to a method for the diagnostic exclusion of the risk of developing relapses in juvenile idiopathic arthritis in a patient, comprising the following steps:

(a) testing of a sample from said patient with a reagent or reagents for the detection of S100A12 amount or concentration and with a reagent or reagent for the detection of hsCRP amount or concentration and

(b) the evaluating the values of the amounts or concentrations measured in step wherein

when the measured value of S100A12 is below a pre-defined cut off value and the measured value of hsCRP is below a pre-defined cut off value, the patient is considered in stable remission or when the measured value of S100A12 is equal or above a pre-defined cut off value and/or the measured value of hsCRP is equal or above a pre-defined cut off value, the patient is considered in instable remission.

[0040] In the methods of the invention one or more additional parameters useful in the assessment of the risk of arthritis relapse can be optionally detected and/or measured. For example, in addition to S100A12 and hsCRP, MRP8/14, ESR or cytokines such as TNF or IL-6 can be detected and/or measured.

[0041] For example, the methods of the invention in addition to the testing of S100A12 and hsCRP MRP8/14 is also/further tested and the test results are all together evaluated.

[0042] Hence, when the measured value of S100A12 is below a pre-defined cut off value, the measured value of hsCRP is below a pre-defined cut off value and the measured value of MRP8/14 is below a pre-defined cut off value there is an indication of low risk of arthritis relapse. A patient is therefore considered in stable remission.

[0043] Hence, when the measured value of S100A12 is equal or greater than a pre-defined cut off value and/or the measured value of hsCRP is equal or greater than a pre-defined cut off value and/or the measured value of MRP8/14 is equal or greater than a pre-defined cut off value, there is an indication of a high risk of arthritis relapse.

[0044] MRP8/14 can be tested with hsCRP and optionally with S100A12, preferably it is measured with hsCRP and S100A12 and the results evaluated all together.

[0045] Alternatively, when the values of S100A12 and hsCRP in a patient are found to be less than the respective predefined cut off value, MRP8/14 can be tested. If the measured values of MRP8/14 is less than the corresponding cut off value then this is a further confirmation that there is a low risk of arthritis relapses and that the patient can be considered in stable remission.

[0046] Hence, in an aspect the present invention is directed to a method for the diagnostic exclusion of the risk of developing relapse in juvenile idiopathic arthritis in a patient comprising the following steps:

(a) testing of a sample from said patient with assay for the detection of S100A12 amount or concentration and with an assay for the detection of hsC P amount or concentration and

(b) evaluating the values of the amounts or concentrations measured in step (a). wherein if the measured value of S100A12 is below a pre-defined cut off value and the measured value of hsCRP is below a pre-defined cut off value, further steps c) and d) are performed c) testing the MRP8/14 amount and/or concentration with an assay for the detection of MRP8/14 d) evaluating the values of the amounts or concentrations wherein if the MRP8/14 amount or concentration is below a predefined cut-off value, stable remission is further confirmed.

[0047] In all the above aspects of the methods of the invention S100A12 and hsCRP are preferably detected and/or measured in a patient that is experiencing arthritis or more preferably that has experienced arthritis or even more preferably that is in arthritis remission on medication. Preferably, the combined tests according to the methods of the invention are performed on sample(s) of a patient in remission on medication to assess the risk of arthritis relapse, preferably of arthritis relapse off medication and therefore to determine whether or not the drug therapy can be withdrawn. Additionally MRP8/14 can be detected.

[0048] Subsequently, the status of the patient with respect to S100A12 and hsCRP and optionally MRP8/14 can be monitored. A patient off medication can be monitored to confirm the stable remission while a patient in remission on medication can be monitored to determine whether or not a stable remission has been achieved.

[0049] In all the aspects of the methods of the invention, S100A12 and hsCRP are preferably periodically monitored after the patient has reached clinical remission on medication. Additionally MRP8/14 an be detected. The monitoring preferably allows the physician to assess the risk of arthritis relapse off medication. Therefore, the combined tests allow the physician to decide whether to withdraw drug therapy or to continue it. A patient off medication can be periodically monitored to confirm the stable remission while a patient in remission on medication can be periodically monitored to determine whether or not a stable remission has been achieved. [0050] Hence, S100A12 and hCP and optionally MRP8/14 are periodically tested in a sample of a patient in clinical remission on medication or off medication or in a patient in stable remission on medication or off medication. Preferably, the patient is monitored with a periodicity of every month, every two months every three months, every six months, every year. For example a patient in stable remission off medication can be monitored for a periodicity of one or two or three months for the year after the drug withdrawal. After one year, the periodicity can be changed to 6 months or one year.

[0051] The methods of the invention are useful in determining the risk of arthritis relapse in a patient, preferably the methods of the invention are useful in determining the risk of arthritis relapses within 3 months to one year after therapy withdrawal, more preferably within 3 months after therapy withdrawal, even more preferably within 6 months after therapy withdrawal, even more preferably within 3 to 6 months after therapy withdrawal.

[0052] In any aspect of the methods of the invention the reagents for the detection of S 100A12, for the detection MRP8/14 and for the detection of hsCRP independently of each other may be reagents for mass spectrometry

[0053] Other preferred embodiments of the present invention are the methods for the diagnostic exclusion of the risk of developing of arthritis relapse in a patient as described above wherein S100A12 is tested applying a cut-off-value at about or at 175 ng/ml. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with an Elisa assay. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured in serum samples. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with and ELISA assay in serum samples. More preferably, the ELISA is as disclosed in WO03/069341 which is herein incorporated by reference in its entirety. In particular in WO03/069341 the preparation of S10012 therein referred to as CALGRANULIN C and the preparation of anti CALGRANULIN C antisera, the determination of CALGRANULIN C by sandwich ELISA are disclosed.

[0054] Other preferred embodiments of the present invention are the methods for the diagnostic exclusion of the risk of developing arthritis relapse in a patient as described above wherein hsCRP is tested in applying a cut-off-value at about or at at 0.3 mg/dl. Preferably, the cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry. Preferably, the cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry in serum samples.

[0055] Other even more preferred embodiments of the present invention are the methods for the diagnostic exclusion of the risk of developing arthritis relapse in a patient as described above wherein S100A12 is tested for applying a cut-off-value at about or at 175 ng/ml and hsCRP is tested for applying a cut-off-value at about or at 0.3 mg/dl. Preferably, the cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry. Preferably, the cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry in serum samples. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with an Elisa assay. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured in serum samples. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with and ELISA assay in serum samples.

[0056] Other preferred embodiments of the present invention are the methods for the diagnosis of arthritis relapse in a patient as described above wherein S100A12 is tested applying a cut-off-value at about or at 175 ng/ml. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with an Elisa assay. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured in serum samples. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with and ELISA assay in serum samples. More preferably, the cut off values is determines as disclosed in the example below using an ELISA assay as disclosed in the below examples and in patent application WO03/069341.

[0057] Other preferred embodiments of the present invention are the methods for the diagnosis of arthritis relapses in a patient as described above wherein hsCRP is tested applying a cut-off-value at about or at 0.3 mg/dl. The cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry. Preferably, the cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry in serum samples.

[0058] Even more preferred embodiments of the present invention are the methods for the diagnosis of arthritis relapses in a patient as described above wherein S100A12 is tested for applying a cut-off-value at about or at 175 ng/ml and hsCRP is tested for applying a cut-off-value at about or at 0.3 mg/dl. Preferably, the cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry. Preferably, the cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry in serum samples. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with an Elisa assay. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured in serum samples. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with and ELISA assay in serum samples. More preferably, the cut off value of 175 ng/ml is determines as disclosed in the example below using an ELISA assay as disclosed in the below examples and in patent application WO03/069341.

[0059] The cut off value for MRP8/14 is preferably at about or at 690 ng/ml. Preferably, the cut off value for MRP8/14 is 690 ng/ml when measured with an Elisa assay. Preferably, the cut off value for MRP8/14 is 690 ng/ml when measured with an Elisa assay in serum samples.

[0060] Suitable cut off values may be defined and determined by those skilled in the art individually for the S100A12, hsCRP and MRP8/14 assay based on known procedure.

[0061] The present invention is directed to a method for the diagnosis of risk of JIA relapse in juvenile idiopathic arthritis (JIA) in a patient, comprising the following steps:

(a) testing of a sample from said patient with a reagent or reagents for the detection of S100A12 amount or concentration and with a reagents or reagents for the detection of hsCRP amount or concentration, and

(b) evaluating the values of the amounts or concentrations measured in step (a).

[0062] Preferably, in the method of the invention when a measured value of S100A12 is below a predefined cut off value and when a measured value of hsCRP is below a pre-defined cut off value there is an indication of a decreased risk of JIA relapse or of a low or lower risk of JIA relapse. Preferably, the risk of JIA relapse is equal or below 30% i.e. the patient has 30% or less of probability of JIA relapse off medication. More preferably, the risk of experiencing JIA relapse is equal or less than 25%. i.e., the patient has 25% or less of probability of JIA relapse off medication. Preferably, a patient having a low risk of JIA relapses according to the invention is considered in stable remission.

[0063] Preferably, when a measured value of S100A12 is equal or above a pre-defined cut off value and/or when the measured value of hsC P is equal or above a pre-defined cut off value there is an indication of an increased risk of JIA relapse or of high or a higher risk of JIA relapse. The risk of JIA relapse can be above 70%, more preferably above 75% i.e., the patient has a probability of 70% or 75% or more of experiencing JIA relapse off medication. Preferably, a patient having a high risk of JIA relapses according to the invention is considered in instable remission.

[0064] Hence, in an aspect the invention is directed to a method for the diagnostic risk of relapses in juvenile idiopathic arthritis (JIA) in a patient, comprising the following steps:

(a) testing of a sample from said patient with a reagent or reagents for the detection of S100A12 amount or concentration and with a hsCRP reagents or reagents for the detection of hsCRP amount or concentration and

(b) evaluating the values of the amounts or concentrations measured in step (a)

i) wherein when the measured value of S100A12 is below a pre-defined cut off value and the measured value of hsCRP is below a pre-defined cut off value, there is an indication of a low risk of JIA relapse or

ii) wherein when the measured value of S100A12 and/or the measured value of hsCRP is equal or above a pre-defined cut off value there is an indication of a high risk of JIA relapse.

[0065] A low or lower risk of JIA relapse is associated with a stable remission in a patient. It is also associated with an immunological remission of the patient. Preferably, the risk of JIA relapse is equal or below 30% i.e. the patient has 30% or less of of probability of JIA relapse off medication. More preferably, the risk of experiencing JIA relapse is equal or less than 25%. i.e., the patient has 25% or less of probability of JIA relapse off medication. Preferably, a patient having a low risk of JIA relapses according to the invention is considered in stable remission.

[0066] A high or higher risk of JIA relapse is associated with an instable remission in a patient. It is also associated with a non-immunological remission of the patient. Preferably, a high or higher risk of experiencing JIA relapse is a risk equal or greater than 70%, preferably equal or greater than 75% of JIA relapse, preferably of JIA relapse off medication i.e., the patient has a probability equal or greater than 70% or 75% to experience JIA relapse preferably off medication. Preferably, a patient having a high risk of JIA relapses according to the invention is considered in instable remission. [0067] In an aspect, the present invention is directed to a method for the diagnosis of risk of relapses in juvenile idiopathic arthritis (JIA) in a patient, comprising the following steps:

(a) testing of a sample from said patient with a reagent or reagents for the detection of S100A12 amount or concentration and with a hsC P reagents or reagents for the detection of hsCRP amount or concentration and

(b) evaluating the values of the amounts or concentrations measured in step (a) wherein

(i) when the measured value of S100A12 is below a pre-defined cut off value and the measured value of hsCRP is below a pre-defined cut off value there is a risk of JIA relapse lower than the risk of JIA relapse of when a measured value of S100A12 and/or a measured value of hsCRP is (are) equal or above a respective pre-defined cut off value or

(ii) when the measured value of S100A12 and/or the measured value of hsCRP is (are) equal above a respective pre-defined cut off value, there is a risk of JIA relapse greater than the ri of JIA relapse of when

a measured value of S100A12 and a measured value of hsCRP are below a respective pre-defined cut off value.

[0068] The hazard ratio (HR) characteristic of the combined tests can be used to determine how high is the risk of JIA relapses of one patient or of a group of patients (e.g., the patient or the group of patients with a S100A12 value and/or a hsCRP value above the respective cut off) with respect to a patient or a group of patients with both S100A12 value and hsCRP value below the respective cut off.

[0069] Hence, in an aspect in the methods of the invention, when the measured value of S100A12 is below a pre-defined cut off value and the measured value of hsCRP is below a pre-defined cut off value there is a risk of JIA relapse about three times lower than the risk of JIA relapse of when a measured value of S100A12 and/or a measured value of hsCRP is (are) equal or above a respective pre-defined cut off value. In an aspect of the invention, when the measured value of S100A12 and/or the measured value of hsCRP is (are) equal or above a respective pre-defined cut off value, there is a risk of JIA relapse about three times greater than the risk of JIA relapse of when the measured value of S100A12 is below a pre-defined cut off value and the measured value of hsCRP is below a predefined cut off value.

[0070] In an aspect, the present invention is directed to a method for the diagnostic exclusion of the risk of developing relapse in juvenile idiopathic arthritis (JIA) in a patient, comprising the following steps: (a) testing of a sample from said patient with a reagent or reagents for the detection of S100A12 amount or concentration and with a reagent or reagent for the detection of hsCRP amount or concentration and

(b) evaluating the values of the amounts or concentrations measured in step (a).

[0071] Preferably, the measured value of S100A12 amount or concentration and the measured value of hsCRP amount or concentration both below the respective pre-defined cut off values are indicative of stable remission.

[0072] Preferably, the measured values of S100A12 and/or of hsCRP amount or concentration, one or both above or equal to the respective pre-defined cut off values are indicative of instable remission.

[0073] Hence, in an aspect the present invention is directed to a method for the diagnostic exclusion of the risk of developing relapses in juvenile idiopathic arthritis in a patient, comprising the following steps:

(a) testing of a sample from said patient with a reagent or reagents for the detection of S100A12 amount or concentration and with a reagent or reagent for the detection of hsCRP amount or concentration and

(b) the evaluating the values of the amounts or concentrations measured in step (a) wherein

when the measured value of S100A12 is below a pre-defined cut off value and the measured value of hsCRP is below a pre-defined cut off value, the patient is considered in stable remission or when the measured value of S100A12 is equal or above a pre-defined cut off value and/or the measured value of hsCRP is equal or above a pre-defined cut off value, the patient is considered in instable remission.

[0074] In the methods of the invention one or more additional parameters useful in the assesment of the risk of JIA relapses can be optionally detected and/or measured. For example in addition to S100A12 and hsCRP, MRP8/14, ESR or cytochines such as TNF or IL-6 can be detected and/or measured.

[0075] For example, the methods of the invention in addition to the testing of S100A12 and hsCRP MRP8/14 is also/further tested and the test results are all together evaluated. [0076] Hence, when the measured value of S100A12 is below a pre-defined cut off value, the measured value of hsCRP is below a pre-defined cut off value and the measured value of MRP8/14 is below a pre-defined cut off value there is an indication of low risk of JIA relapse. A patient is therefore considered in stable remission.

[0077] Hence, when the measured value of S100A12 is equal or greater than a pre-defined cut off value and/or the measured value of hsCRP is equal or greater than a pre-defined cut off value and/or the measured value of MRP8/14 is equal or greater than a pre-defined cut off value, there is an indication of a high risk of JIA relapse.

[0078] MRP8/14 can be tested with hsCRP and optionally with S100A12, preferably it is measured with hsCRP and S100A12 and the results evaluated all together.

[0079] Alternatively, when the values of S100A12 and hsCRP in a patient are found to be less than the respective predefined cut off value, MRP8/14 can be tested. If the measured values of MRP8/14 is less than the corresponding cut off value then this is a further confirmation that there is a low risk of JIA relapses and that the patient can be considered in stable remission.

[0080] Hence, in an aspect the present invention is directed to a method for the diagnostic exclusion of the risk of developing relapse in juvenile idiopathic arthritis (JIA) in a patient comprising the following steps:

(a) testing of a sample from said patient with assay for the detection of S100A12 amount or concentration and with an assay for the detection of hsCRP amount or concentration and

(b) evaluating the values of the amounts or concentrations measured in step (a). wherein if the measured value of S100A12 is below a pre-defined cut off value and the measured value of hsCRP is below a pre-defined cut off value, further steps c) and d) are performed c) testing the MRP8/14 amount and/or concentration with an assay for the detection of MRP8/14 d) evaluating the values of the amounts or concentrations wherein if the MRP8/14 amount or concentration is below a predefined cut-off value, stable remission is further confirmed.

[0081] In all the aspects of the methods of the invention S100A12 and hsCRP are preferably detected and/or measured in a patient that is experiencing JIA or more preferably that has experienced JIA or even more preferably that is in JIA remission on medication. Preferably, the combined tests according to the methods of the invention are performed on sample(s) of a patient in remission on medication to assess the risk of JIA relapse, preferably of JIA relapse off medication and therefore to determine whether or not the drug therapy can be withdrawn. Additionally MRP8/14 can be detected. [0082] Subsequently, the status of the patient with respect to S100A12 and hsCRP and optionally MRP8/14 can be monitored. A patient off medication can be monitored to confirm the stable remission while a patient in remission on medication can be monitored to determine whether or not a stable remission has been achieved.

[0083] In all the aspects of the methods of the invention, S100A12 and hsCRP are preferably periodically monitored after the patient has reached clinical remission on medication. Additionally MRP8/14 an be detected. The monitoring preferably allows the physician to assess the risk of JIA relapse off medication. Therefore, the combined tests allow the physician to decide whether to withdraw drug therapy or to continue it. A patient off medication can be periodically monitored to confirm the stable remission while a patient in remission on medication can be periodically monitored to determine whether or not a stable remission has been achieved.

[0084] Hence, S100A12 and hCPR and optionally MRP8/14 are periodically tested in a sample of a patient in clinical remission on medication or off medication or in a patient in stable remission on medication or off medication. Preferably, the patient is monitored with a periodicity of every month, every two months every three months, every six months, every year. For example a patient in stable remission off medication can be monitored for a periodicity of one or two or three months for the year after the drug withdrawal. After one year, the periodicity can be changed to 6 months or one year.

[0085] The methods of the invention are useful in determining the risk of JIA relapse in a patient, preferably the methods of the invention are useful in determining the risk of JIA relapses within 3 months to one year after therapy withdrawal after therapy withdrawal, more preferably within 3 months after therapy withdrawal, even more preferably within 6 months after therapy withdrawal, even more preferably within 3 to 6 months after therapy withdrawal.

[0086] In any aspect of the methods of the invention, the detection of S100A12 and of hsCRP in the sample may occur simultaneously or sequentially.

[0087] In any aspect of the methods of the invention, the reagents for the detection of S100A12, for the detection MRP8/14 and for the detection of hsCRP independently of each other are preferably based on test principles selected from the following group: ELISA, turbidimetry, nephelometry, particle enhanced turbidimetry, particle enhanced nephelometry, latex agglutination, lateral flow assays, and mass spectrometry. More preferably the reagents are for Elisa assay, turbidimetry and nephelometry.

[0088] In any aspect of the methods of the invention the reagents for the detection of S 100A12, for the detection MRP8/14 and for the detection of hsCRP independently of each other may be reagents for immunoassays.

[0089] In any aspect of the methods of the invention the reagents for the detection of S 100A12, for the detection MRP8/14 and for the detection of hsCRP independently of each other may be reagents for mass spectrometry

[0090] In the methods of the invention, the sample is a biological sample from a patient. Preferably, the biological sample is selected from serum, plasma or whole blood. [0091] In the methods of the invention, S100A12 and hsC P may be assayed for using different immunoassay test principles known to the skilled person such as turbidimetry, nephelometry, particle enhanced turbidimetry, particle enhanced nephelometry, latex agglutination, ELISA and all other conceivable immunochemical test methods.

[0092] Suitable cut-off values may be defined by those skilled in the art individually for the S100A12 assay and the hsCRP assay based on well-known procedures.

[0093] Other preferred embodiments of the present invention are the methods for the diagnostic exclusion of the risk of developing of JIA relapse in a patient as described above wherein S100A12 is tested applying a cut-off-value at about or at 175 ng/ml. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with an Elisa assay. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured in serum samples. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with and ELISA assay in serum samples. More preferably, the cut off value of 175 ng/ml is determines as disclosed in the examples below using an ELISA assay as disclosed in the below examples and in patent application WO03/069341.

[0094] Other preferred embodiments of the present invention are the methods for the diagnostic exclusion of the risk of developing JIA relapse in a patient as described above wherein hsCRP is tested in applying a cut-off-value at about or at at 0.3 mg/dl. Preferably, the cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry. Preferably, the cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry in serum samples.

[0095] Other even more preferred embodiments of the present invention are the methods for the diagnostic exclusion of the risk of developing JIA relapse in a patient as described above wherein S100A12 is tested for applying a cut-off-value at about or atl75 ng/ml and hsCRP is tested for applying a cut-off-value at about or at 0.3 mg/dl. Preferably, the cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry. Preferably, the cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry in serum samples. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with an Elisa assay. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured in serum samples. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with and ELISA assay in serum samples. More preferably, the cut off value of 175 ng/ml is determines as disclosed in the example below using an ELISA assay as disclosed in the below examples and in patent application WO03/069341.

[0096] Other preferred embodiments of the present invention are the methods for the diagnosis of JIA relapse in a patient as described above wherein S100A12 is tested applying a cut-off-value at about or atl75 ng/ml. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with an Elisa assay. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured in serum samples. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with and ELISA assay in serum samples. More preferably, the cut off value of 175 ng/ml is determines as disclosed in the example below using an ELISA assay as disclosed in the below examples and in patent application WO03/069341.

[0097] Other preferred embodiments of the present invention are the methods for the diagnosis of relapses of juvenile rheumatoid arthritis in a patient as described above wherein hsCRP is tested applying a cut-off-value at about or at 0.3 mg/dl. The cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry. Preferably, the cut off value for hsC P is 0.3 mg/dl when measured in nephelometry in serum samples.

[0098] Even more preferred embodiments of the present invention are the methods for the diagnosis of relapses of juvenile rheumatoid arthritis in a patient as described above wherein S100A12 is tested for applying a cut-off-value at about or at 175 ng/ml and hsCRP is tested for applying a cut-off-value at about or at 0.3 mg/dl. Preferably, the cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry. Preferably, the cut off value for hsCRP is 0.3 mg/dl when measured in nephelometry in serum samples. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with an Elisa assay. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured in serum samples. Preferably, the cut-off value is of 175 ng/ml when S100A12 is measured with and ELISA assay in serum samples. More preferably, the cut off value of 175 ng/ml is determines as disclosed in the example below using an ELISA assay as disclosed in the below examples and in patent application WO03/069341.

[0099] The cut off value for MRP8/14 is preferably at about or at 690 ng/ml. Preferably, the cut off value for MRP8/14 is 690 ng/ml when measured with an Elisa assay. Preferably, the cut off value for MRP8/14 is 690 ng/ml when measured with an Elisa assay in serum samples.

[0100] Suitable cut off values may be defined and determined by those skilled in the art individually for the S100A12, hsCRP and MRP8/14 assay based on known procedure.

[0101] The present invention is further directed to kit useful in the methods of the invention. The kit contains reagents for the detection and/or measurement of S100A12 and hsCRP amount and/or concentrations. The Kit may optionally contain the reagent for the detection of MRP8/14.

[0102] The present invention is further directed to drugs for the treatment and/or prevention of JIA relapse in a patient at risk of JIA relapses, the risk preferably, as determined by the method of the invention. Preferably the drug is for the prevention of JIA relapse. The patient preferably has a high risk of JIA relapse as determined by the method of the inventions. More preferably, the high risk of JIA relapse is of equal of above the 70% and even more preferably above the 75% as determined by the methods of the inventions. The drugs are preferably selected from the group of nonsteroidal- antiinflammatory drugs, corticosteroids, immunosuppressive agents, and biological, more preferably the drug is methotrexate.

[0103] In all the methods of the invention as disclosed above MRP8/14 may be measured in place of S100A12.

Detailed description of the invention.

[0104] The invention shall now be further described by the following examples with respect to the attached figures. All examples are provided by way of example only, without any intended limitation of the scope of the invention. All cited references are incorporated herein by reference in their entireties.

[0105] In the figures:

[0106] Figure 1 shows the biomarker levels of patients who experienced a flare within 0-3, 3-6, and 6- 12 months and of those with stable remission over one year. The upper and lower bounds of each box indicate the 25th and 75th percentile, respectively; heavy lines within the box, the median; whiskers are drawn to the nearest value not beyond a standard span from the 25th and 75th percentile, respectively, with the standard span being defined as 1.5 times (interquartile range); values beyond the whiskers (outliers) are drawn individually. P-values report a comparison of patients who experienced a flare within 0-3/3-6/6-12 months with patients in remission over at least 3/6/12 months after discontinuing medication (Mann-Whitney U test).

[0107] Figure 2 shows Kaplan-Meier curves of the time from the biomarker analysis to disease flare. In particular the graphs show a follow-up of 12 months after withdrawal of therapy, which was the time when the biomarker analysis was performed. The y-axis shows the proportion of patients without flares after discontinuing medication.

[0108] Figure 3 shows the proposed stratification of patients in remission. Patients with JIA in clinical remission have an elevated risk of flares after withdrawal of therapy if subclinical inflammation is present. This would be a status of clinical but not immunological remission. Since S100A12 (plus hsC P) may detect subclinical disease, a stratification of patients according to their immunological status is feasible. Adapting the criteria for JIA remission by Wallace et al. the methods of the invention allow to base the decision to either stop therapy or continue with drugs that maintain clinical remission on a stratification based upon the biomarker result. In cases with continued therapy, the maintenance therapy may contribute to immunological remission after a while. Thus, with biomarker analyses the stratification may be repeated over time with proposed intervals of 3 months. Red lines represent phases with active disease, while green lines represent phases with inactive disease.

[0109] Figure 4 is a scheme of the distribution of standards and probe in the S100A12 ELISA determination.

[0110] The term "clinical remission" means remission wherein only the clinical criteria for remission are met. Clinical remission without immunological remission is also defined as "instable remission". Immunological remission is measured according to the methods of the invention. For example high S100A12 and/or hsCRP (i.e. above or equal to the pre-defined cut off values) are indicative of absence of immunological remission and therefore in the presence of a clinical remission are indicative of "instable remission".

[0111] The term "stable remission" means remission wherein both clinical and immunological remissions are observed. For example low S100A12 and hsCRP (i.e. below the pre-defined cut off values) are indicative of immunological remission and therefore in the presence of a clinical remission are indicative of stable remission. Patients in remission from arthritis e.g. JIA already have a background risk of experiencing relapses. The present invention allows determining whether a patient in remission on medication has a risk of developing relapses lower or grater that the background risk of flares typical of arthritis e.g. JIA in case of therapy withdrawal.

[0112] The terms "hsCRP reagent or reagents" or "reagent or reagents for the detection of hsCRP" or similar expressions refer to the use of assays that allow the determination of concentration and/or amount of CRP below the detection limit of standard CRP assay. For the specific purpose hsCRP assays are used. [0113] The term "hsC P value" or similar indicates that the CRP has been measured with hsCRP assays. Hence "CRP" measured with an hsCRP assay is referred to as "hsCRP".

[0114] The term "arthritis" includes any form of arthritis, for example, juvenile idiopathic arthritis (J IA) rheumatoid arthritis, psoriatric arthritis or ankylosing arthritis. The preferred form of arthritis according to the present invention is J IA.

[0115] The term "Juvenile idiopathic arthritis" (JIA) indicates a chronic disease characterized by relapsing joint inflammation in children. Subtypes of J IA can be for example oligoarticular, polyarticular, and systemic J IA, for a total of seven subtypes.

[0116] The term "relapse" indicates the return of signs and symptoms of a disease after a remission. It can be on or off medication. The methods of the invention determine the risk associated with arthritis relapse, preferably of J IA relapse. Preferably the method determines the risk of relapse off medication.

[0117] The term "S100A12" refers to a neutrophil activation marker known in the art. It is also referred to as "Calgranulin C" or "Calgranulin C polypetide".

[0118] ELISA assay for detecting S100A12 in a biological sample is disclosed for example in WO03/069341.

Example 1: S100A12 Elisa assay [0119] l. Primry antibody:

1 plate (Nunc Maxisorp) was coated as following: Coating 16μΙ a-S100A12 (lmg/ml) + 5 ml of carbonate buffer at pH 9,5. (corresponding to 160ng/well) were incubated overnight at 4 °C or at 37 °C for 1 h. It has been observed that incubation overnight at 4 °C performs better. The wells were than washed 3 times with wash buffer

[0120] 2. Blocking phase

ΙΟΟμΙ blocking buffer /well were used in the blocking phase; the wells were incubated for one hour at 1 h 20 °C and then washed once with wash buffer

[0121] 3.Antigen:

Standard: 12μΙ of S100A12 stoch solution+ 638μΙ block buffer for a dilution factor of 1:54, 16 were prepared corresponding to 250ng/ml. Further dilutions with a factor 1:2 were performed to 0.25ng/ml according to the following dilution scheme

250ng/ml ^ l25ng/ml ^64ng/ml ^32ng/ml ^ l6ng/ml^8ng/ml^4ng/ml ^2ng/ml ^ lng/ml ^0,5ng /ml ->0,25ng/ml.

Samples: diluted in blocking buffer twice at 1:2 and further diluted 1:1, 1:2,1:4 each dilution twice Standard and samples: were used starting at A2 (see fig. 4) (with 0.25 ng/mL ST) [0122] For each standard and probe a double determination has been performed (row 1 will remain free = BLAN K); the wells were incubated for 2 h at 20 °C and then the wells were washed 3 times with wash buffer per 200μΙ/ννθΙΙ.

[0123] 4. Biotinylierter-S100A12 Antibody:

7.9 μΙ (X-S100A12- Bio +6.25 ml of blocking buffer were mixed. of this solution was used with an incubation of 30 min at 37 ° C; then the wells were washed 3 times with wash buffer

[0124] 5.Streptavidin peroxidase:

3.13 μΙ Streptavidin-peroxidase +6.25 ml of wash buffer were prepared. (row 1 remain free) of this solution was used with an incubation of 30 min at 37 ° C incubation; then the wells were washed 3 times with wash buffer

[0125] 6.Substrat:

0.01 g of ABTS + pH4 + 10μΙ 25ml of Citratebuffer and 10μΙ H202 solution were prepared and protected from light in aluminum foil. were used. Measurement occurred after 20 min at 405nm.

[0126] The buffers used were:

Wash buffer: PBS +0.02% Tween

Blocking buffer: PBS +0.02% +0.25% Tween BSA

Carbonate buffer: 0.1 M NaHCO 3 +0.1 M Na2Co3 -^pH 9,5

Citrate buffer: 0.05 M citrate +0.1 M Na2H P04 ->pH4, 0

[0127] Example 2: Identification of the combination S100A12 and hsCRP for the determination of the J IA relapses risk.

a) Patients enrolled

[0128] 364 enrolled patients were enrolled in a clinical trial disclosed in JAMA 2010;303:1266-73. Of these 364 enrolled patient serum samples were available for 188 (52%) patients which were used by the present inventors to arrive at the methods of the invention. The serum samples were collected and the biomarkers S100A12, MRP18/14 and hsCRP were analyzed at the time of therapy withdrawal, i.e. confirmed clinical remission on medication.

[0129] More in particular, in the controlled trial (JAMA 2010;303:1266-73), JIA patients with inactive disease for at least 3 months were randomised to withdraw all medication after further 6 or 12 months of continuous inactive disease status. Thus in total, study patients' inactive disease status continued for 6+3=9 or 12+3=15 months before medication was withdrawn, respectively. The remission in this JAMA study was confirmed clinically based on the physicians evaluation of disease activity, the absence of systemic signs/symptoms, of active arthritis and of uveitis, and by erythrocyte sedimentation rate (ES ) as well as standard CRP.

b) Laboratory measurements

[0130] Serum concentrations of MRP8/14 and S100A12 were determined by sandwich enzyme-linked immunosorbent assays as previously reported, according to the procedure disclosed in Foell et al. Phagocyte-specific calcium-binding S100 proteins as clinical laboratory markers of inflammation. Clin Chim Acta 2004; 344(l-2):37-51. In particular, S100A12 is measured with an enzyme-linked immunosorbent assay (ELISA) as disclosed in example 1 and in patent application WO03/069341 which is incorporated herein by reference.

[0131] Samples were analyzed once the patient in clinical remission, at the time when all antiinflammatory therapies were stopped. The analyzing laboratory was blinded for the patients' characteristics. A hsCRP test measuring low levels of CRP using laser nephelometry was applied.

[0132] The biomarker levels were compared between different patient groups using the nonparametric Mann-Whitney U test. The comparison between patients who experienced a flare within 6 months and patients with stable remission over at least 6 months after therapy withdrawal constitutes the primary statistical analysis that provides confirmatory statistical evidence. Moreover, ROC analyses were performed in order to evaluate the predictive value of all biomarkers for the risk of disease flares. All biomarkers were dichotomized applying optimal cut-off levels as per ROC analysis.

[0133] Specifically, cut-off levels were measured so that predicting flares within 6 months after therapy withdrawal a maximal Youden-lndex was provided (According to Youden WJ. Index for rating diagnostic tests. Cancer 1950;3(l):32-5 herein incorporated by reference). The time from therapy withdrawal to disease flare in a maximum 12-month follow-up period was evaluated by univariate and multivariate methods for survival data. Patients without flare within 12 months were censored and accounted to be in stable remission.

[0134] Based on a proportional hazards model, the hazard ratio (HR) of each molecular biomarker was estimated. Point estimates are provided with 95% confidence intervals (CI). P-values are considered statistically significant in the case of p<0.05. Statistical analyses were performed using SAS (Version 9.2 for Windows, SAS Institute Inc., Cary, NC, USA). The study complied with the Standards for Reporting of Diagnostic Accuracy (STARD) guidelines. c) Results

1. Overall performance of the biomarkers

[0135] Of 188 enrolled patients, 120 remained in clinical remission over one year while 15, 20, and 33 (68 in total) patients experienced a flare within 0-3, 3-6, and 6-12 months after therapy withdrawal, respectively. Median follow-up of all 188 patients was 25.7 months from therapy withdrawal (mean 28.8, range 0.5-67.7 months), and median follow-up of those 120 patients who remained in stable remission over one year was 25.7 months from therapy withdrawal (mean 28.8, range 11.7-67.7 months). [0136] Comparing patients who stopped therapy at six months versus twelve months after randomisation, there were no relevant differences in mean and median biomarker values. Similarly, both patient groups did not differ relevantly with respect to the flare rate within three months after therapy withdrawal. Clinical or standard laboratory parameters could not differentiate between patients at risk for relapse and those without.

[0137] Comparing patients who experienced a flare within 6 months to those with stable remission after therapy withdrawal, the former had significantly higher S100A12 concentrations (median 130 versus 68 ng/ml, p=0.0023 significantly higher MRP8/14 concentrations (median 850 versus 440 ng/ml, p=0.0061), but no significantly different hsCRP levels (median 0.04 versus 0.04 mg/dl, p=0.1215).

[0138] Figure 1 shows the median biomarker levels of patients who experienced a flare within 0-3, 3- 6, 6-12 months and of those with stable remission over one year after therapy withdrawal. Particularly high levels of S100A12 and MRP8/14 were associated with early flares within 0-3 months after the biomarker analysis. Later flares within 3-6 months and 6-12 months after therapy withdrawal were also associated with elevated levels of S100A12 and MRP8/14. These markers, analyzed at the time when stopping therapy in remission, indicate subclinical inflammatory activity, i.e. a status of unstable remission with a high risk of disease flares within 3 months.

[0139] A monotone relation of biomarker levels and time to disease flare exists: with increasing time to disease flare median S100A12 and MRP8/14 levels were monotonically decreasing. These consistent results indicate that the observed association with the flare risk represents a valid and reliable finding. However, in case of the biomarker hsCRP no such consistent results have been observed.

[0140] In ROC analyses the above results regarding the high association of the biomarkers S100A12 and MRP8/14 with the flare risk as well as the lower association of hsCRP were confirmed. Differentiating flares within 6 months versus stable remission over at least 6 months after therapy withdrawal, the resulting areas under the ROC curves of S100A12, MRP8/14 and hsCRP were 0.6600 (CI 0.5502-0.7698), 0.6455 (CI 0.5360-0.7550) and 0.5845 (CI 0.4679-0.7010), respectively. The corresponding figures of flares within 3 months were 0.6987 (CI 0.5324-0.8650), 0.7510 (CI 0.5991- 0.9030) and 0.4697 (CI 0.2681-0.6712), respectively.

2. Performance of dichotomized biomarkers

[0141] The measured cut-off levels of the biomarkers S100A12, MRP8/14 and hsCRP for optimal prediction of disease flares within 6 months after therapy withdrawal were 175 ng/ml, 690 ng/ml and 0.3 mg/dl, respectively. Applying these cut-off levels, the Youden-lndex of the biomarkers S100A12, MRP8/14 and hsCRP of predicting disease flares within 6 months was 0.3134, 0.2822 and 0.2183, respectively.

[0142] Table 1 shows further diagnostic statistics of the dichotomized biomarkers. The biomarkers S100A12 and MRP8/14 perform particularly well predicting early flares within 3 months after therapy withdrawal. In contrast, the biomarker hsCRP may add some information predicting later flares. A combined marker, i.e. a predictive algorithm that results from a logic combination of the single markers is thus shown. [0143] Specifically, the predicted outcome of the combined marker "S100A12 or hsCRP" was set positive if S100A12≥175ng/ml or hsCRP≥0.3mg/dl.

[0144] As shown in Table 1, the combined marker "S100A12 or hsCRP" brings together the different specific predictive advantages of the single biomarkers and attained the best predictive performance. Hence it outperforms all single biomarkers with respect to the prediction of early as well as later flares.

Table 1. Diagnostic statistics of dichotomized biomarkers

S100A12 or MRP8/14 or

S100A12 M P8/14 hsCRP

hsCRP hsCRP

S100A12 >175 MRP8/14 ≥690

Positive predicted outcome if ...

≥175 ng/ml >690 ng/ml >0.3 mg/dl ng/ml or hsCRP ng/ml or (cut-off level)*

0.3 mg/dl hsCRP >0.3 mg/dl

Flare within 0-3 months after therapy

withdrawal

Sensitivity %, (95% CI) 67 (38-88) 87 (60-98) 29 (8-58) 79 (49-95) 93 (66-100)

Specificity %, (95% CI) 84 (77-89) 64 (57-71) 87 (68-81) 75 (68-81) 58 (50-66)

PPV %, (95% CI) 26 (13-43) 17 (10-28) 15 (4-34) 20 (11-34) 15 (8-25)

NPV %, (95% CI) 97 (92-99) 98 (94-100) 94 (89-97) 98 (94-100) 99 (95-100)

Positive likelihood ratio 4.12 2.42 2.14 3.14 2.22

Negative likelihood ratio 0.40 0.21 0.82 0.29 0.12

Youden-lndex 0.5048 0.5083 0.1520 0.5357 0.5100

Flare within 3-6 months after therapy

withdrawal

Sensitivity %, (95% CI) 30 (12-54) 45 (23-68) 35 (15-59) 55 (32-77) 65 (41-85)

Specificity %, (95% CI) 86 (79-91) 65 (57-73) 89 (83-94) 79 (72-85) 61 (53-69)

PPV %, (95% CI) 21 (8-41) 15 (7-26) 30 (13-53) 26 (14-41) 18 (10-29)

NPV %, (95% CI) 90 (84-95) 90 (83-95) 91 (86-95) 93 (87-97) 93 (86-97)

Positive likelihood ratio 2.09 1.30 3.33 2.61 1.67

Negative likelihood ratio 0.82 0.84 0.73 0.57 0.57

Youden-lndex 0.1562 0.1036 0.2447 0.3395 0.2618

Flare within 0-6 months after therapy

withdrawal

Sensitivity %, (95% CI) 46 (29-63) 63 (45-79) 32 (17-51) 65 (46-80) 76 (59-89)

Specificity %, (95% CI) 86 (79-91) 65 (57-73) 89 (83-94) 79 (72-85) 61 (53-69)

PPV** %, (95% CI) 42 (26-59) 29 (19-41) 41 (22-61) 41 (28-55) 31 (21-42)

NPV** %, (95% CI) 87 (81-92) 88 (81-94) 86 (79-91) 91 (85-95) 92 (85-97)

Positive likelihood ratio 3.18 1.81 3.07 3.07 1.97

Negative likelihood ratio 0.63 0.57 0.76 0.45 0.38

Youden-lndex 0.3134 0.2822 0.2183 0.4365 0.3766

* The predicted outcome of the single biomarkers was set positive if the observed level was greater or equal than the reported cut- level, respectively.

** PPV, positive predictive value; NPV, negative predictive value.

[0145] Table 2 shows the number of flares of patients with high and low values of the dichotomized biomarkers at different timepoints after therapy withdrawal.

[0146] In the above approaches the predictive performance of a (single or combined) marker is evaluated assigning equal weights to sensitivity and specificity. Instead one may search for a marker with especially high sensitivity (accepting a lower specificity), or vice versa.

[0147] In Table 3, three successively connected strategies are evaluated, in that the decision to stop or continue therapy is based on (i) the single marker S100A12 (ii) the combination "S100A12 or hsCRP", and (iii) a further combination of all markers "S100A12 or hsCRP or MRP8/14".

* Two patients with missing hsCRP result have been excluded.

[0148] If combined marker sets are used, the percentage of a wrong decision to "stop therapy" is highly reduced.

[0149] In univariate survival analyses the time from therapy withdrawal to disease flare in a maximally 12 month follow-up period was evaluated. For S100A12, the hazard ratio of flare was HR=2.81 (CI 1.70-4.65, p<0.0001). For MRP8/14 it was HR=2.24 (CI 1.39-3.62, p=0.0009) and for hsCRP HR=2.25 (CI 1.27-4.00, p=0.0058).

[0150] To illustrate the predictive performance of the biomarkers S100A12 and hsCRP, Figure 2 shows Kaplan-Meier curves of the time from therapy withdrawal to disease flare of patients with high versus low S100A12 and hsCRP values, respectively. The corresponding Kaplan-Meier curve for MRP8/14 has been published previously. Beyond univariate survival analyses, in a multivariate model all three biomarkers were included simultaneously.

[0151] Multivariate analyses revealed that in the presence of S100A12 and hsCRP, MRP8/14 did not provide useful additional information for the prediction of the time to disease flare (p=0.1049). For the best combined marker "S100A12 or hsCRP" the resulting hazard ratio of flare was HR=2.96 (CI 1.83-4.79, p<0.0001). [0152] In further multivariate survival analyses, beyond the molecular biomarkers S100A12, M P8/14 and hsCRP, the demographic and clinical variables gender, age at disease onset, disease subtype (persistent oligoarthritis, polyarticular group, systemic JIA) and methotrexate therapy (6 versus 12 months after induction of disease remission) were included. The above results were confirmed qualitative.

[0153] S100A12, MRP8/14 and hsCRP are meaningful for prediction the relapse risk in JIA. The present inventors have found that each of the single phagocyte activation markers S100A12 and MRP8/14 provides good diagnostic performance in the prediction of disease flares, if they are used separately. Both biomarkers are especially useful in predicting early flares within 3 months after therapy withdrawal. The acute phase reactant hsCRP provides some independent additional information on later flares. Without being bound to any theory, this may be the reason why the predictive performance of the marker S100A12 is improved by adding hsCRP. The best combined marker panel (as shown by the best Youden-lndex in table 1) is "S100A12 and/or hsCRP".

Application of the methods of the invention

[0154] There is a clear need to stratify JIA patients according to their risk profile and the potential course of the disease. Therapeutic stratification is currently performed on the basis of disease categories and severity of inflammation. However, these approaches focus on the initiation of therapies. There is no doubt that physicians also need guidance at times of low disease activity or after reaching remission. It is a key task to adapt the intensity of anti-inflammatory therapy in order to balance the need for sufficient control of disease activity and the potential harm of therapeutics in the developing and growing child. With this regard physicians also need to stratify patients for the need of a maintenance therapy versus the option to stop all medications. In an attempt to allow approaches to patients who are successfully treated, an international consensus on JIA remission was achieved. According to these preliminary remission criteria, 6 continuous months of inactive disease on medication defines clinical remission on medication. It is unclear whether therapy should be continued in remission on medication for a variable time before stopping.

[0155] The present inventors have shown that biomarkers of inflammation can be used to stratify patients at this point. In the presence of low phagocyte activation markers, the likelihood of a flare is low even after stopping therapy. On the other hand, subclinical disease activity may result in instable remission, e.g. a status of clinical but not immunologic remission. As biomarkers of inflammation, phagocyte activation markers from the group of calcium-binding S100 proteins (plus hsCRP) have a close correlation to actual disease activity and therefore detect subclinical inflammation at the time of the analysis, if present. Applied as snapshot analysis, the biomarkers inform about the stability of remission and the risk of flares, especially within a timeframe of a few weeks up to 3 months. This is also reflected by the drop of patients in remission at around 3 months after the biomarker test (Figure 2).

[0156] While on the one hand with the determination of subclinical activity the flare risk over the following 3 months may be assessed, the patient is also protected to some extent for another 6-12 weeks after therapy withdrawal due to the long half-life of some drugs used in the pharmaceutical treatment of JIA (e.g., methotrexate). [0157] Therefore, the drop in patients with a higher risk profile due to subclinical disease activity at around 3 months after stopping e.g. methotrexate could be explained by the fact that at this time the drug is no longer effective in preventing the flare, leading to a peak of relapses in patients with instable disease remission around this time point.

[0158] It is therefore meaningful to repeat the analysis at intervals of approximately 3 months to monitor the immunological status of JIA patients (Figure 3).

[0159] In conclusion, the neutrophil activation marker S100A12 and/or the phagocyte activation marker M P8/14 supplemented by hsCRP indicate subclinical inflammation at the molecular level and thereby identify patients at risk of relapse, even if clinical signs of disease activity are absent. In particular, they allow excluding the risk of relapse in high percentage. Molecular markers of inflammation can be used to stratify patients at times of low disease activity and to optimize strategies of personalized medicine.