Protein diagnostic biomarkers for severe drug eruption

Abstract

To develop biomarkers for diagnosis of the pathology, exacerbation, and type of severe drug eruptions such as drug-induced hypersensitivity syndrome and Stevens-Johnson syndrome / toxic epidermal necrolysis. A test method for severe drug eruption that includes measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from a subject.

Description

Technical Field

[0001] The present invention relates to a method for assisting in the diagnosis of the onset and disease severity of severe drug eruptions.

Background Art

[0002] Severe drug eruptions such as Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and drug-induced hypersensitivity syndrome (DIHS) can leave serious sequelae or even lead to death. Therefore, it is important to diagnose them early and promptly initiate appropriate treatment. For this reason, biomarkers that can diagnose DIHS and SJS / TEN at an early stage are in demand.

[0003] To date, while sufficient verification is required for blood protein markers of severe drug eruptions, TARC (chemokine CCL17), granulysin (GNLY), FAS-L, interleukin 6 (IL-6), IP-10 (chemokine CXCL10), and combinations thereof have been proposed (Non-Patent Documents 1, 2, 3). Stratifin (SFN), also known as 14-3-3 sigma, is a protein with a molecular weight of 27,000. An increase in the expression of SFN has been suggested to be associated with carcinogenesis in lung tissue (Non-Patent Document 4). In addition, in skin tissue, its gene expression is induced by ultraviolet exposure, and a technique for examining the degree of skin damage using this as an index has been developed (Patent Document 1).

[0004] CD30 (TNFRSF8) is one of the members of the TNF receptor superfamily and is a type I transmembrane protein with a molecular weight of 120 kDa. It is known to be expressed in activated lymphocytes in healthy individuals and strongly expressed in tumor cells of Hodgkin lymphoma in hematological malignancies. Therefore, the examination of CD30 by tissue immunological techniques has attracted attention as being useful for the diagnosis of Hodgkin lymphoma. Recently, its expression has also been confirmed in anaplastic large cell lymphoma and non-hematological tumors, and anti-CD30 antibody therapy and the like have also been developed (Non-Patent Document 5). However, until now, it has been unclear whether SFN and soluble CD30 present in the blood can detect SJS / TEN and DIHS, and what their effects are. In addition, regarding interleukin-1 receptor antagonist (IL-1Ra) and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), their association with the onset of severe drug eruptions was not known.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Patent Document

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a method for assisting in diagnosing Stevens-Johnson syndrome / toxic epidermal necrolysis (SJS / TEN) and drug hypersensitivity syndrome (DIHS) with high accuracy, a method for objectively evaluating the therapeutic efficacy of these diseases, and a method for assisting in differentiating between mild drug eruption cases that heal without progression and cases that progress to severe forms.

Means for Solving the Problems

[0008] From the plasma proteome measurement results of severe drug eruption patients by the SOMAscan method, biomarker proteins considered useful for diagnosing the exacerbation or disease progression of Stevens-Johnson syndrome / toxic epidermal necrolysis (SJS / TEN) and drug hypersensitivity syndrome (DIHS) were explored. Among the measured values of 1310 proteins, proteins showing significant fluctuations in the acute phase of these severe drug eruptions were explored. In addition to the previously reported TARC (chemokine CCL17) and granulysin (GNLY), four proteins, stratifin (SFN, 14-3-3 protein sigma), TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) were identified. As a result of verification by ELISA, the protein with the highest ability to diagnose the disease state of DIHS (discrimination between the acute phase and the recovery phase) and the ability to discriminate exacerbation (discrimination from mild drug eruption) was CD30, showing better results than the previously reported granulysin and TARC. In addition, in the diagnosis of the disease state and exacerbation of SJS / TEN, SFN showed the best biomarker performance. In addition, by combining these proteins, the positive rates of DIHS and SJS / TEN can be improved, and SJS / TEN and DIHS with different treatment regimens can be well distinguished. In particular, the use of the SFN / TARC ratio was useful for the specific diagnosis of SJS / TEN (differential diagnosis from DIHS).

[0009] The present invention has been completed based on these findings. The gist of the present invention is as follows. (1) A method for examining severe drug eruption, comprising measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from a subject. (2) The method according to (1), further comprising measuring the expression of at least one protein selected from the group consisting of granulysin (GNLY) and CC motif chemokine 17 (TARC / CCL17) in a sample derived from a subject. (3) The method according to (1) or (2), wherein the protein whose expression is measured is at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), and the measured value aids in the diagnosis of severe exacerbation of severe drug eruption. (4) The method according to (1) or (2), wherein the protein whose expression is measured is at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), and the measured value aids in the diagnosis of the disease trend of severe drug eruption. (5) The method according to (1) or (2), wherein the protein whose expression is measured is stratifin, and the measured value aids in the diagnosis of drug hypersensitivity syndrome and / or Stevens-Johnson syndrome / toxic epidermal necrolysis. (6) The method according to (1) or (2), wherein the protein whose expression is measured is TNF receptor superfamily member 8 (CD30 / TNFRSF8), and the measured value aids in the diagnosis of drug hypersensitivity syndrome and / or Stevens-Johnson syndrome / toxic epidermal necrolysis. (7) The method according to (1) or (2), wherein the protein whose expression is measured is at least one combination of proteins selected from the group consisting of the combination of stratifin and CC motif chemokine 17 (TARC / CCL17) and the combination of stratifin and TNF receptor superfamily member 8 (CD30 / TNFRSF8), and the measured value aids in the specific diagnosis of drug hypersensitivity syndrome or Stevens-Johnson syndrome / toxic epidermal necrolysis. (8) A method for diagnosing severe drug eruption, comprising: a3. Obtaining a sample from a subject; b3. Measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from the subject; and c3. Determining the severity of the drug eruption based on the measurement value of b3. The method as described above. (9) A method for diagnosing severe drug eruption, comprising: a2. Obtaining a sample from a subject; b2. Measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from the subject; and c2. Determining the progression of the severe drug eruption based on the measurement value of b2. The method as described above. (10) A method for diagnosing severe drug eruption, comprising: a4. Obtaining a sample from a subject; b4. Measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from the subject; and c4. Determining the type of the severe drug eruption based on the measurement value of b4. The method as described above. (11) A method for diagnosing and treating severe drug eruption, comprising: a3. Obtaining a sample from a subject; b3. Measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from a subject, c3. Determining the severity of drug eruption based on the measurement value of b3, and d3. Discontinuing the administration of a suspected drug that a subject who has developed or is highly likely to develop severe drug eruption is taking, and initiating appropriate treatment according to the severity of the drug eruption The method as described above including the above. (12) A method for diagnosing and treating severe drug eruption, a2. Obtaining a sample from a subject, b2. Measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from a subject, c2. Determining the disease state of severe drug eruption based on the measurement value of b2, and d2. Discontinuing the administration of a suspected drug that a subject who is highly likely to be in the acute stage of severe drug eruption is taking, and initiating appropriate treatment The method as described above including the above. (13) A method for diagnosing and treating severe drug eruption, a4. Obtaining a sample from a subject, b4. Measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from a subject, c4. Determining the disease type of severe drug eruption based on the measurement value of b4, and d4. Discontinuing the administration of the suspected drug being taken by the subject diagnosed with the disease type of severe drug eruption, and initiating appropriate treatment according to the disease type of severe drug eruption The method as described above. (14) A kit for the examination of severe drug eruption, including a reagent capable of measuring the expression in a sample derived from a subject for at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B).

Effect of the Invention

[0010] It is most important to initiate treatment for severe drug eruption as early as possible. The proteins discovered by the present inventors serve as new blood markers (severe disease discrimination markers) for differentiating between mild patients and those with DIHS or SJS / TEN, and further as markers useful for diagnosing the disease state (disease state diagnosis markers), such as whether patients with DIHS or SJS / TEN have entered the recovery phase. They also serve as markers (disease type determination markers) useful for determining whether the disease type of the patient is DIHS or SJS / TEN. In particular, stratifin and CD30 show higher accuracy than existing marker candidates such as TARC and granulysin, and by measuring in combination with existing ones such as TARC and granulysin, early detection and specific diagnosis of patients with DIHS or SJS / TEN become possible. This specification includes the content described in the specification and / or drawings of Japanese Patent Application No. 2020-73955, which is the basis of the priority of this application.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Mode for Carrying Out the Invention

[0012] Hereinafter, embodiments of the present invention will be described in detail.

[0013] The present invention provides a method for examining severe drug eruptions, which includes measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from a subject.

[0014] Drug rash is a rash that occurs as a reaction to drugs and is a type of side effect. Generally, it often develops 1 to 2 weeks after taking the drug and can be alleviated by discontinuing the causative drug, so the diagnosis is easy. However, severe drug rash, which is a serious drug rash, often does not improve only by discontinuing the causative drug, and if appropriate treatment is not carried out, it can threaten life, so early diagnosis is important.

[0015] Stevens-Johnson syndrome (SJS) and toxic epidermal necrosis (TEN), which are representative examples of severe drug rash, are intractable diseases with a high fatality rate and sequelae such as blindness and respiratory disorders. SJS and TEN are considered to be in the same spectrum of pathology. Although the symptoms of TEN are similar to those of SJS, they are more severe and often progress from SJS. In SJS, extensive mucosal lesions are seen at the skin-mucosal transition sites such as the lips, conjunctiva, and vulva accompanied by fever, and erosions and blisters based on necrotic damage to the epidermis are characteristic along with generalized erythema of the skin. The area is less than 10% of the total body surface area. On the other hand, TEN shows extensive erythema and significant necrotic damage to the epidermis such as blisters and epidermal detachment over 10% of the body, accompanied by high fever and mucosal rash, and is considered the most severe among skin disorders caused by pharmaceuticals. The incidence of SJS and TEN is reported to be 1 to 6 cases and 0.4 to 1.3 cases per year per million population, respectively.

[0016] Drug-induced hypersensitivity syndrome (DIHS) is also a severe type of drug eruption comparable to SJS / TEN. Erythematous papules and erythema multiforme accompanied by high fever of 38 degrees Celsius or higher are observed throughout the body and progress to erythroderma. Usually, there are no or mild mucosal eruptions, although erosions of the oral mucosa are sometimes recognized. Systemic lymph node swelling, organ damage including liver dysfunction, and peripheral white blood cell abnormalities (leukocytosis, eosinophilia, appearance of atypical lymphocytes) are seen. Reactivation of human herpesvirus (HHV)-6 occurs during the course, and when lymph node swelling or HHV-6 reactivation cannot be proven, it is diagnosed as atypical DIHS. Different from ordinary drug eruptions, it often does not occur immediately after administration of the causative drug but rather after more than two weeks, and the symptoms continue even after discontinuation of the causative drug, often requiring more than one month to resolve.

[0017] Regarding these severe drug eruptions, it is considered important to diagnose them early and promptly initiate appropriate treatment. However, since the initial symptoms are similar to those of mild drug eruptions, it is difficult to identify or predict patients who will deteriorate at an early stage. Also, when treating patients with deteriorating conditions, it is important to know the disease type of the patient early and appropriately grasp the disease severity (activity of the disease state).

[0018] So far, TARC (chemokine CCL17), known as a biomarker for assessing the severity of atopic dermatitis, has been proposed for the verification of DIHS or the detection of SJS / TEN, and proteins such as granulysin and Fas ligand have been proposed as blood protein markers for severe drug eruptions.

[0019] Stratifin, also known as 14-3-3 sigma, is a protein consisting of 248 amino acids encoded by the SFN gene. It is expressed in the epithelium of the esophagus and skin and is known to have increased expression in head and neck cancer, lung cancer, cervical cancer, etc. In the epidermis of the skin, stratifin expression is increased in a p53-dependent manner by ultraviolet rays and is thought to act on dermal fibroblasts and be involved in the control of wound healing and fibrosis. The UniProt number is P31947.

[0020] Tumor necrosis factor receptor superfamily member 8 (CD30 / TNFRSF8) is a type I transmembrane protein belonging to the TNF receptor family with 593 amino acids encoded by the gene TNFRSF8. In healthy adults, its expression is only observed in activated T and B cells, while Hodgkin lymphoma strongly expresses it in mononuclear Hodgkin cells, multinucleated Reed-Sternberg cells, undifferentiated large cell lymphoma, etc. CD30 causes the activation of NFκB by phosphorylating IκB and is involved in the regulation of inflammation, cell survival, and proliferation along with cytokine secretion. In clinical tests, cellular immune tests are performed as markers for adult T-cell leukemia, Hodgkin lymphoma, and Ki-1 lymphoma. Soluble CD30 is reported to be cleaved and released by the binding of CD30L and increase in an oxidative stress state. The UniProt number is P28098.

[0021] Interleukin-1 receptor antagonist (IL-1Ra) is an extracellular secreted protein with 159 amino acids encoded by the IL1RN gene. It binds to the receptor IL-1R1 and inhibits the activation of interleukin-1 alpha and -1 beta. High expression is observed in cells of the bone marrow, immune system, and gastrointestinal tract. In skin tissue, it is known to be involved in regulating skin aging. The UniProt number is P18510.

[0022] Tumor necrosis factor receptor superfamily member 6B (DcR3 / TNFRSF6B), officially registered as tumor necrosis factor receptor superfamily member 6B, is a protein with 245 amino acids encoded by the gene TNFRSF6 and is known as a decoy receptor belonging to the TNF receptor family. By binding to Fas ligand as a decoy receptor, it has the effect of inhibiting Fas-induced apoptosis signals. It is known that blood DcR3 increases in patients with rheumatoid arthritis. The Uniprot number is O95407.

[0023] Stratifin, CD30, IL-1Ra, and DcR3 / TNFRSF6B are novel and useful as biomarkers for severe drug eruption.

[0024] In the method of the present invention, it is preferable that the protein whose expression is measured is stratifin, and the measured value aids in the diagnosis of drug hypersensitivity syndrome (DIHS) and / or Stevens-Johnson syndrome / toxic epidermal necrolysis (SJS / TEN). Diagnosis includes diagnosis of disease severity, diagnosis of exacerbation, specific diagnosis of disease type, and the like.

[0025] Alternatively, in the method of the present invention, it is preferable that the protein whose expression is measured is CD30, and the measured value aids in the diagnosis of DIHS and / or SJS / TEN. Diagnosis includes diagnosis of disease severity, diagnosis of exacerbation, specific diagnosis of disease type, and the like.

[0026] In the method of the present invention, furthermore, for those known as blood protein markers for severe drug eruption such as granulysin (GNLY) and TARC, the expression in a sample derived from a subject may be measured. By combining these novel markers with each other or combining novel markers with known markers, the positive rate of severe drug eruption can be improved and the specificity can be increased.

[0027] In the present invention, the subject is a mammal suspected of developing severe drug eruption, but may be directed to all mammals in which the risk of onset is considered. Typically it is a human. Examples of samples derived from the subject include cells, tissues, body fluids, etc. obtained from the subject, specifically, the subject's blood (e.g., whole blood, serum, plasma, extracorporeal plasma exchange fluid, etc.) and bronchoalveolar lavage fluid. Whole blood, serum or plasma obtained by ordinary blood tests (clinical tests) may be used as the blood sample.

[0028] In the method of the present invention, the measurement of the expression in a sample derived from a subject may be performed by measuring the abundance of the above protein or a fragment thereof in the sample. As the means for measurement, a known method may be used without particular limitation. It is preferable to measure at the protein level, but measurement at the nucleic acid level may also be performed.

[0029] In order to measure the expression of the above protein at the protein level, it is advisable to use an antibody that specifically recognizes the above protein. The antibody may be either a monoclonal antibody or a polyclonal antibody. These antibodies can be produced by known methods, or commercially available ones may be used. Typical methods include immunoassay methods such as the ELISA method and immunochromatography method. Since immunoassay methods do not require special equipment or techniques and can simply and rapidly detect and quantify the target protein, they can also be preferably used for the measurement of the above protein in the present invention. Antibodies against the above protein are known and there are also commercially available products. Further, as described above, since the amino acid sequences of the above proteins and the nucleotide sequences encoding them are also known, specific antibodies against each protein may be prepared by creating general hybridomas.

[0030] Immunoassays themselves are well known in this field. Depending on the reaction format, there are sandwich assays, competitive assays, agglutination assays, Western blot assays, etc., and based on the label, there are enzyme immunoassays, radioimmunoassays, fluorescence immunoassays, luminescence immunoassays, etc. In the present invention, any immunoassay capable of quantitative detection may be used. Although not particularly limited, for example, a sandwich assay such as sandwich ELISA can preferably be used. In a sandwich assay, an antibody that binds to the target protein is immobilized and reacted with a sample. The target protein bound to the immobilized antibody is measured using a detection antibody labeled with an enzyme or the like. It is preferable to use an antibody that binds to the target protein at a site different from the immobilized antibody for the detection antibody. The immobilized antibody and the detection antibody may be polyclonal antibodies or monoclonal antibodies, and antigen-binding fragments of the antibodies can also be used. After reacting the target protein bound to the immobilized antibody with the detection antibody and performing washing, the amount of the bound detection antibody is measured by a signal from the substance labeled on the antibody. For example, when an antibody labeled with alkaline phosphatase is used as the detection antibody, the substrate of the enzyme is added to the reaction system, and the amount of color development, fluorescence, or luminescence generated by the enzyme reaction can be measured with a corresponding device. An immunoassay is performed on a standard sample with a known concentration containing the target protein, and a calibration curve plotting the relationship between the signal of the labeling substance and the concentration is prepared. For a sample with an unknown concentration of the target protein, the same operation is performed, and the obtained signal measurement value is applied to the calibration curve to quantify the target protein in the sample.

[0031] In order to measure the expression of the above protein at the nucleic acid level, it is advisable to use a nucleic acid probe that can specifically hybridize with the mRNA of the above protein (when measuring by Northern blotting). Alternatively, at least one pair of nucleic acid primers that can specifically amplify the cDNA synthesized using the mRNA of the above protein as a template may also be used (when measuring by RT-PCR). The nucleic acid probe and nucleic acid primers can be designed based on the gene information (described above) of the above protein. Usually, a nucleic acid probe of about 15 to 1500 bases is appropriate. The nucleic acid probe may be labeled with a radioactive element, a fluorescent dye, an enzyme, etc. Usually, a nucleic acid primer of about 15 to 30 bases is appropriate. The nucleic acid primer may also be labeled with a radioactive element, a fluorescent dye, an enzyme, etc.

[0032] The protein or gene whose expression is to be measured may be one type or multiple types. By referring to multiple gene expression and multiple protein expression data, more accurate evaluation may be possible. In order to simultaneously detect multiple gene expressions and multiple protein expressions, detection methods such as DNA array (probe fixed on a substrate) (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, DECEMBER 2002, 951-960), protein chip (antibody fixed on a substrate) (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, SEPTEMBER 2002, 683-695), Luminex (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, JUNE 2002, 447-456), etc. may be used.

[0033] Examples of the combination of the protein or gene whose expression is to be measured include, but are not limited to, the combinations and ratios described in Table 10 of the following examples (CD30+TARC, CD30+SFN, CD30+GNLY, CD30+IL-1Ra, SFN / TARC ratio, SFN / CD30 ratio).

[0034] For at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), measure the expression in a sample derived from a subject, and based on the expression level, the severity discrimination, exacerbation discrimination, and disease type discrimination of severe drug eruption can be performed.

[0035] Therefore, the method of the present invention can assist in the diagnosis of severe drug eruption (severity diagnosis, exacerbation diagnosis, specific diagnosis of disease type).

[0036] As an example of the present invention, the diagnosis of severe drug eruption can be performed according to the following criteria. Measure the expression of at least one of the above proteins in the plasma collected from the subject. If a value higher than a preset cut-off value or reference value is obtained, the subject is evaluated as having developed severe drug eruption. This preset cut-off value can be appropriately set by those skilled in the art. For example, the 95% confidence interval of the quantitative value of healthy subjects who have not developed severe drug eruption can be used as the reference value, or the cut-off value can be set from the ROC curve. Alternatively, if at least one of the above proteins shows an upward trend compared to past measurement values, the possibility of developing severe drug eruption is suspected. The cut-off values of each biomarker candidate shown in Tables 5 to 8 of the Examples described below were set with reference to the Youden Index (sensitivity + specificity - 100) by performing ROC curve analysis on the acute phase of DIHS or SJS / TEN and the control groups (total recovery group, mild group, or various skin disease groups).

[0037] The method of the present invention can be used for the diagnosis of severe drug eruption exacerbation. In this specification, "diagnosis of exacerbation" means the discrimination between severe drug eruptions such as SJS / TEN and DIHS, and mild drug eruptions such as disseminated erythematous papular rash and erythema multiforme. When the expression level of at least one protein selected from the group consisting of stratifin, CD30, IL-1Ra, and DcR3 is high in a sample derived from a subject, it can be determined that the subject is likely to develop or is developing severe drug eruption in the future. For example, as the cut-off values for differentiating DIHS, SJS / TEN from mild drug eruptions, stratifin: 2 - 8 ng / mL (preferably 3.7 ng / mL), DcR3: 1.5 - 3 ng / mL (preferably 2.6 ng / mL), IL-1Ra: 1000 - 2000 pg / mL (preferably 1600 pg / mL), CD30: 50 - 150 ng / mL (preferably 82 ng / mL), etc. can be set. The present invention is a method for diagnosing severe drug eruption, comprising: a3. Obtaining a sample from a subject; b3. Measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from the subject; and c3. Determining the severity of the drug eruption based on the measurement value in b3. The present invention provides the above method.

[0038] In addition, the method of the present invention can be used for the diagnosis of the disease state of severe drug eruption. In this specification, "diagnosis of disease state" means the discrimination between the acute phase and the recovery phase of severe drug eruptions such as SJS / TEN and DIHS. When the expression level of at least one protein selected from the group consisting of stratifin, CD30, IL-1Ra, and DcR3 is high in a sample derived from a subject, it can be determined that the subject is likely to be in the acute phase of severe drug eruption. For example, as cut-off values for the diagnosis of the severity of DIHS or SJS / TEN, stratifin can be set at 1 to 3 ng / mL (preferably 1.0 ng / mL), DcR3 at 0.3 to 2 ng / mL (preferably 1.1 ng / mL), IL-1Ra at 500 to 1500 pg / mL (preferably 930 pg / mL), CD30 at 50 - 150 ng / mL (100 ng / mL), and so on. The present invention relates to a method for diagnosing severe drug eruption, comprising: a2. obtaining a sample from a subject; b2. measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from the subject; and c2. determining the severity of the severe drug eruption based on the measurement value in b2. The present invention provides the above method.

[0039] SFN has particularly high detection performance for SJS / TEN, and CD30 has high detection performance for DIHS. DcR3 and IL-1Ra have detection power for both.

[0040] Furthermore, the method of the present invention can be used for the specific diagnosis of severe drug eruptions. As used herein, "specific diagnosis" means the differentiation of the types of severe drug eruptions. Severe drug eruptions such as SJS / TEN and DIHS can sometimes result in serious sequelae or even death. Therefore, the ability to use the method for the specific diagnosis of severe drug eruptions has great clinical significance. For example, when the expression level of stratifin in a sample from a subject is high, it can be determined that the disease type is likely to be SJS / TEN. Also, when the expression level of CD30 in a sample from a subject is high, it can be determined that the disease type is likely to be DIHS. In order to improve the detection rate of severe drug eruptions, biomarkers may be combined. The combination of stratifin and TARC can improve the discriminatory performance for SJS / TEN. When differentiating SJS / TEN, the SFN / TARC ratio may be used. In addition, the combination of CD30 and TARC, the combination of CD30 and stratifin, the combination of CD30 and granulysin (GNLY), and the combination of CD30 and IL-1Ra can improve the detection performance for DIHS. For example, as the cut-off values for the specific diagnosis of SJS / TEN, stratifin: 3 - 7 ng / mL (preferably 4.3 ng / mL), for the specific diagnosis of DIHS, CD30: 60 - 240 ng / mL (preferably 124 ng / mL), DcR3: 1 - 3 ng / mL (preferably 2.6 ng / mL), IL-1Ra: 1000 - 4000 pg / mL (preferably 2040 pg / mL), etc. can be set. In order to improve the detection performance for DIHS and SJS / TEN, each marker can also be used in combination at these cut-off values. Also, the detection performance for DIHS and SJS / TEN can be improved by combining with previously reported TARC (cut-off value: 5500 pg / mL as an example) and GNLY (cut-off value: 10.7 ng / mL as an example). The cut-off value of the SFN / TARC ratio for improving the discriminatory performance of SJS / TEN can be set to 4 - 7 (preferably 5.6). The present invention is a method for diagnosing severe drug eruptions, comprising a4. obtaining a sample from a subject, b4. Measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from a subject, and c4. Determining the disease type of severe drug eruption based on the measured value of b4 Providing the method comprising the above.

[0041] The method of the present invention can be used not only for the diagnosis of the disease state of severe drug eruption but also for the prognosis examination and confirmation of the treatment effect. For example, for at least one protein selected from the group consisting of stratifin, CD30, IL-1Ra, and DcR3, the expression in a sample derived from a subject determined to be highly likely to be in the acute stage of exacerbation of severe drug eruption is measured once or multiple times at different times. When the expression level decreases to a level close to the cut-off value or reference value, it is determined that the subject has recovered from severe drug eruption by treatment. When the level is high or does not decrease, it can be determined that the subject has not recovered from severe drug eruption by treatment or the recovery is insufficient. The cut-off value for determining recovery from treatment is valid as the cut-off value for disease state diagnosis.

[0042] If it is determined that the subject has developed, or is highly likely to have developed, severe drug eruption, the administration of the suspected drug should be discontinued, and appropriate treatment should be initiated according to the symptoms of the patient's disease severity and disease type. As drug therapy for DIHS, systemic steroid administration (starting from 0.5 - 1 mg / kg / day in terms of prednisolone equivalent and gradually reducing as appropriate) is effective, and caution should be exercised when reducing the dose due to recurrence of symptoms caused by reactivation of HHV-6. In the case of SJS / TEN, systemic steroid administration with different doses according to severity (in terms of prednisolone equivalent, 0.5 - 1 mg / kg / day for moderate cases, 1 - 2 mg / kg / day for severe cases, and starting from 1 g / day of methylprednisolone for 3 days for the most severe cases, and gradually reducing as appropriate according to symptoms) is recommended. In addition, high-dose human immunoglobulin (IVIG) intravenous therapy (administering 400 mg / kg / day continuously for 5 days, usually only 1 course), plasmapheresis, treatment of ocular surface inflammation for eye lesions (instilling betamethasone or dexamethasone about 4 times a day), preventing symblepharon to preserve the ocular surface epithelium, and preventing infections of the ocular surface, etc. can be carried out.

[0043] In the United States, severe drug eruptions are also important side effects. Regarding SJS / TEN, the "Genetic and Rare Diseases Information Center (GARD)", established with funding from the National Institutes of Health (NIH), has published information on symptoms, causes, treatment methods, and prognosis (https: / / rarediseases.info.nih.gov / diseases / 7700 / stevens-johnson-syndrometoxic-epidermal-necrolysis). Treatments include the need for hospitalization and discontinuation of the administered medications, as well as supportive therapy and drug therapy. Supportive therapy generally involves treating skin symptoms similar to those of severe burns, including wound care, pain management, fluid and electrolyte management, nutritional support, body temperature management, and monitoring or treatment of secondary infections. For eye symptoms, irrigation with saline is available. Regarding drug therapy, systemic corticosteroids, intravenous immunoglobulin (IVIG), cyclosporine, plasmapheresis, and anti-tumor necrosis factor (TNF) monoclonal antibodies are mentioned, but it is stated that none have been sufficiently studied in randomized trials. Regarding ophthalmic drug therapy, multiple daily lubrications with preservative-free eye drops or ointments and ophthalmic drug therapy including topical corticosteroid drugs and broad-spectrum antibiotics are mentioned. Additionally, in a 2018 survey of 22 university hospitals, among the treatments for 377 SJS / TEN patients, only supportive therapy accounted for 29.3%, only steroids accounted for 30.0%, IVIG therapy accounted for 24.9%, and both steroid and IVIG therapy accounted for 14.3% (Michaeletti RG et al., J Invest Dermatol., 2018; 138: 2315-2321).

[0044] Regarding DIHS (also known as Drug Reaction with Eosinophilia and Systemic Symptoms, DRESS), although symptoms and clinical studies are described in GARD, there is no description of treatment methods (https: / / rarediseases.info.nih.gov / diseases / 13629 / drug-reaction-with-eosinophilia-and-systemic-symptoms). In an analysis report using the electronic medical information of Partners HealthCare System in Boston, out of 3,162,562 patients, 69 had DRESS, and corticosteroids were administered to 51 patients. Among them, topical corticosteroids were used in 35 patients, oral corticosteroids in 36 patients, and intravenous corticosteroids in 26 patients (Wolfson AR et al., J Allergy Clin Immunol Pract., 2019;7:633-640)

[0045] The present invention relates to a method for diagnosing and treating severe drug eruption, comprising: a3. Obtaining a sample from a subject; b3. Measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from the subject; c3. Determining the severity of the drug eruption based on the measurement value of b3; and d3. Discontinuing the administration of a suspected drug that the subject with severe drug eruption has developed or is likely to develop, and starting appropriate treatment according to the severity of the drug eruption The present invention provides the above method. In addition, the present invention relates to a method for diagnosing and treating severe drug eruption, comprising: a2. Obtaining a sample from a subject; b2. Measuring the expression in a sample derived from a subject for at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), c2. Determining the severity of severe drug eruption based on the measurement value of b2, and d2. Discontinuing the administration of the suspected drug taken by the subject determined to be highly likely to be in the acute stage of severe drug eruption and initiating appropriate treatment Providing the method comprising the above. Furthermore, the present invention is a method for diagnosing and treating severe drug eruption, a4. Obtaining a sample from a subject, b4. Measuring the expression in a sample derived from a subject for at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), c4. Determining the disease type of severe drug eruption based on the measurement value of b4, and d4. Discontinuing the administration of the suspected drug taken by the subject for whom the disease type of severe drug eruption has been determined and initiating appropriate treatment according to the disease type of severe drug eruption Providing the method comprising the above.

[0046] The present invention also provides a kit for the examination of severe drug eruption, which comprises a reagent capable of measuring the expression in a sample derived from a subject for at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B).

[0047] As an example, the kit of the present invention includes, as a reagent, an antibody that can specifically recognize the above protein. The antibody may be immobilized on a microtiter plate, magnetic beads, a cellulose membrane, or a substrate. The kit may further include an instrument for collecting a sample from a subject, an anticoagulant, a set of reagents for detecting the above protein, an instruction manual, and the like. The instruction manual may describe, in addition to the method of using the kit, the evaluation and / or discrimination criteria for the acute stage of severe drug eruption

[0048] As another example, the kit of the present invention includes, as a reagent, a nucleic acid probe that can specifically hybridize with the mRNA of the above protein. The nucleic acid probe may be immobilized on a substrate. The kit may further include an instrument for collecting a biological sample, an anticoagulant, reagents for extracting RNA from a sample derived from a subject, reagents for detecting RNA, an instruction manual, and the like. The instruction manual may describe, in addition to the method of using the kit, the evaluation and / or discrimination criteria for the acute stage of severe drug eruption

[0049] As yet another example, the kit of the present invention includes, as a reagent, at least one pair of nucleic acid primers that can specifically amplify cDNA synthesized using the mRNA of the above protein as a template. The kit may further include an instrument for collecting a sample from a subject, an anticoagulant, reagents for extracting RNA from a sample derived from a subject, reagents for detecting RNA, an instruction manual, and the like. The instruction manual may describe, in addition to the method of using the kit, the evaluation and / or discrimination criteria for the acute stage of severe drug eruption

[0050] In addition to the above, the kit of the present invention may further include a standard protein, a buffer, a substrate (when the antibody is enzyme-labeled), a reaction stop solution, a washing solution, a reaction container, and the like.

[0051] The kit of the present invention can also be used for the diagnosis of SJS caused by infectious diseases such as mycoplasma, in addition to severe drug eruption, and can be used as a pharmaceutical for diagnosing diseases.

Example

[0052] The present invention will be described in more detail below with reference to examples. [Example 1] (1) Specimen Regarding the severe drug eruption specimens used for analysis, they were collected with the approval of the research ethics committees of each affiliated hospital, the National Institute of Health Sciences, the Kihara Foundation, Astellas Pharma, and Daiichi Sankyo. At Yokohama City University, Shimane University, Shimada City Hospital, Iwata City General Hospital, Nara Medical University, and Niigata University, blood samples were taken from patients suspected of having mild drug eruptions (erythema multiforme, disseminated erythematous papular rash, eczematous rash) and related skin diseases. In addition, patients with skin diseases other than drug eruptions (atopic dermatitis, psoriasis, autoimmune blistering diseases, infectious skin diseases (excluding measles, rubella, and chickenpox)) were also collected, and their plasma and serum were cryopreserved at -80°C. Specimens from healthy individuals collected by NIES Co., Ltd. were used.

[0053] The breakdown of the specimens is shown in Table 1. 75 specimens of onset cases and 12 specimens of healthy individuals collected by November 2017 were used for the search by SOMAscan analysis and the verification of the ELISA kit (Discovery-set, Table 1). Next, as an independent separate group sample different from the Discovery-set, specimens collected after December 2017 (163 specimens of onset cases and 38 specimens of healthy individuals not used in the search) were used for the verification of biomarkers (Validation set). Furthermore, the biomarker performance was evaluated using the Combined-set obtained by combining both the Discovery-set and the Validation-set and adding related diseases.

[0054] JPEG0007705847000001.jpg102146

[0055] (2) Search for biomarker candidates Using proteomic analysis with SomaLogic's SOMAscan system, we searched for biomarkers for Stevens-Johnson syndrome / toxic epidermal necrolysis (SJS / TEN) and drug-induced hypersensitivity syndrome (DIHS), which are clinically important severe drug eruption types. The SOMAscan system is a measurement method that detects 1,310 proteins using aptamers (artificial ligands made of single-stranded nucleic acids). Plasma specimens in the discovery set were sent frozen to SomaLogic, Inc. in the United States for SOMAscan measurement.

[0056] Based on the SOMAscan measurement values of 1,310 proteins, we searched for (1) biomarker candidates capable of discriminating the disease severity of DIHS and SJS / TEN (comparison between the acute stage of exacerbation and the recovery stage), and (2) severity-related biomarker candidates that can be discriminated from the mild group (comparison between the acute stage of exacerbation and the mild group). After logarithmic transformation of the measurement values (fluorescence signal intensity) of each probe, the samples were divided into a mild drug eruption group (Mild, n = 29), a DIHS acute stage group (DHIS, n = 14), an SJS / TEN acute stage group (n = 9), a recovery stage group of all cases (All-R, n = 19), and a healthy control group (HC, n = 12). Focusing on proteins whose effect size Hedge's g value and / or mean fold change (FC) were within the top 10 and showed an AUC value of 0.85 or more in ROC curve analysis in either the acute stage group of DIHS or SJS / TEN, we found that it included stratifin, CD30, DcR3, and IL-1Ra, which had not been known to be useful as severe drug eruption biomarkers, along with TARC and granulysin (GNLY), which are known as reported severe drug eruption biomarkers. Table 2 shows these biomarker candidates and the ELISA measurement system used in subsequent verification experiments.

[0057] JPEG0007705847000002.jpg48161

[0058] (3) Comparison of measurement results Figure 1 shows the fluctuation patterns of the SOMAscan and ELISA measurement values ​​of six types of biomarker candidates. In the SOMAscan analysis, a significant increase was observed in each type of severe drug rash compared to the healthy group and the mild drug rash group for all proteins, and the level in the recovery group decreased to a level close to that of the healthy group. Similar results were obtained with ELISA, and the concentration ranges of the healthy group, the increase ranges in the acute phase of DIHS and SJS / TEN cases, and the concentration ranges of the total recovery group in the Discovery-set and Validation-set were similar (Figure 1).

[0059] (4) Usefulness in diagnosing disease progression and progression 4-1) Evaluation using the Discovery set Table 3 shows the results of comparing the disease severity discrimination ability (distinguishing between acute and recovery phases of severe disease) and severity discrimination ability (distinguishing between severe and mild drug rash groups) of each candidate protein found in the Discovery-set for DIHS and SJS / TEN, based on the AUC values ​​calculated from ROC curve analysis. SFN, DcR3, and IL-1Ra were predicted to have high discrimination ability for both DIHS and SJS / TEN, and SFN showed particularly high detection ability for SJS / TEN. In contrast, CD30 had high detection ability for DIHS, and its disease severity discrimination ability (AUC 0.93) and severity diagnosis ability (AUC 0.87) were higher than those of the previously reported TARC (AUC 0.89 and 0.82).

[0060] JPEG0007705847000003.jpg541700.85 and above are shown in bold.

[0061] 4-2) Validation using validation-set Table 4 shows the results of the ROC analysis of the Validation-set. Similar to the Discovery-set, in the Validation-set, SFN showed high discriminative performance especially for SJS / TEN, CD30 showed high discriminative performance for DIHS, and DcR3 and IL-1Ra showed discriminative performance for both. When comparing the disease severity discrimination ability and the severe disease diagnosis ability of DIHS, DcR3 (AUC 0.99 and 0.86) and CD30 (AUC 0.94 and 0.89) exceeded the previously reported TARC (AUC 0.91 and 0.78) and GNLY (AUC 0.86 and 0.81). In terms of the disease severity discrimination ability of SJS / TEN, SFN (AUC 0.92) showed the highest value. The severe disease discrimination ability of SFN for SJS / TEN seen in the Discovery-set (AUC 0.91, Table 3) decreased slightly in the Validation-set (AUC 0.79), but in the Combined-set combining the Discovery-set and the Validation-set, SFN was the highest (AUC 0.84), followed by IL-1Ra (AUC 0.80), which was higher than the previously reported GNLY (AUC 0.76) (Table 4).

[0062] The numerical values of JPEG0007705847000004.jpg 651700.85 or more are shown in bold.

[0063] 4-3) Evaluation results by the Combined-set 4-3-1) Disease specificity Figure 2 shows the results of comparing the distribution of measured values of each candidate protein in various skin diseases including drug eruption using the data of the Combined-set. A strong increase was observed for all candidate proteins in DIHS and / or SJS / TEN. In particular, SFN showed an increase characteristic of SJS / TEN, while in other disease types including DIHS and EM-major, or autoimmune bullous diseases, it only showed a moderate increase. On the other hand, CD30 showed a DIHS-specific increase similar to TARC, and a slight increase was also observed in a part of EM-major, but there was almost no increase in autoimmune bullous diseases. Different from SFN and CD30, DcR3 and GNLY tended to increase in both DIHS and SJS / TEN.

[0064] 4-3-2) Discrimination of the disease severity of DIHS and SJS / TEN Table 5 shows the analysis results of the data of the Combined-set and indicates the disease severity discrimination performance of each candidate protein for DIHS and SJS / TEN. Cutoff values were set to distinguish the acute phase and the total recovery period (All-R) of DIHS and SJS / TEN, and the sensitivity and specificity were compared. In the discrimination of the disease severity of DIHS, DcR3 (AUC 0.96) and CD30 (AUC 0.94) were excellent. When the cutoff value of DcR3 was 1.1 ng / mL and that of CD30 was 100 ng / mL, both had a sensitivity of 80% or more and a specificity of 90% or more, and discriminated the acute phase of DIHS from the total recovery period group. DcR3 and SFN were excellent in the disease severity discrimination performance of SJS / TEN (both AUCs were 0.9 or more). When the cutoff value of DcR3 was 0.55 ng / mL, the sensitivity was 90% or more and the specificity was 85% or more. When the cutoff value of SFN was 1.0 ng / mL, both the sensitivity and specificity were 80% or more, and the acute phase of SJS / TEN was discriminated from the total recovery period.

[0065] From the results of the ROC curve analysis when comparing the acute phase and overall recovery period (All-R) of JPEG0007705847000005.jpg63170 DIHS and SJS / TEN, the concentration at which the Youden Index was highest with a specificity of 80% or more was set as the cut-off value. Numerical values with an AUC of 0.85 or more are shown in bold. Cut-off values were not evaluated for proteins with a discrimination performance of AUC 0.7 or less.

[0066] 4-3-3) Discrimination of disease severity in DIHS and SJS / TEN Table 6 shows the analysis results of the Combined-set data and indicates the disease severity discrimination performance of each candidate protein in DIHS and SJS / TEN. Cut-off values were set for the discrimination between the acute phase and the mild group (Mild) of DIHS and SJS / TEN, and sensitivity and specificity were compared. In the discrimination of disease severity in DIHS, CD30 (AUC 0.88) was the most excellent, being higher than the previously reported GNLY (AUC 0.85) and TARC (AUC 0.79). When the cut-off value of CD30 was set at 82 ng / mL, the sensitivity and specificity were both 80% or more (Table 6). On the other hand, in the discrimination of disease severity in SJS / TEN, SFN was the most excellent (AUC 0.84). When the cut-off value was 3.7 ng / mL, the sensitivity was approximately 70% and the specificity was approximately 90% for differentiating the acute phase and the mild group of SJS / TEN.

[0067] From the results of the ROC curve analysis when comparing the acute phase and the mild group of JPEG0007705847000006.jpg63170 DIHS and SJS / TEN, the concentration at which the Youden Index was highest with a specificity of 80% or more was set as the cut-off value. Numerical values of sensitivity and specificity of 80% or more with an AUC of 0.8 or more are shown in bold. Cut-off values were not evaluated for proteins with a discrimination performance of AUC 0.7 or less.

[0068] 4-3-4) Specific diagnosis of the disease type of severe drug eruption Among severe drug eruptions, SJS / TEN and DIHS are particularly severe disease types. Since their treatment strategies are different, it is important to diagnose early whether it is SJS / TEN or DIHS. Therefore, the performance of detecting DIHS (DIHS-specific diagnostic performance) and the performance of specifically detecting SJS / TEN (SJS / TEN-specific diagnostic performance) were compared among various skin diseases of each protein.

[0069] Table 7 shows the results of setting cut-off values suitable for the specific diagnosis of DIHS and SJS / TEN and comparing sensitivity and specificity from the comparison of DIHS and other skin diseases (including mild drug eruptions, severe drug eruptions other than DIHS, and other skin diseases), and the comparison of SJS / TEN and other skin diseases (including mild drug eruptions, severe drug eruptions other than SJS / TEN, and other skin diseases). In the specific diagnostic performance of DIHS, CD30 is the most excellent (AUC 0.87). When the cut-off value is 124 ng / mL, both the sensitivity and specificity are around 80%. In the specific diagnostic performance of SJS / TEN, SFN is the highest (AUC 0.79). When the cut-off value is 4.3 ng / mL, the sensitivity is 60.9% and the specificity is 81.8%.

[0070] From the results of ROC curve analysis when comparing DIHS and other skin diseases (including mild drug eruptions, severe drug eruptions other than DIHS, and other skin diseases), and SJS / TEN and other skin diseases (including mild drug eruptions, severe drug eruptions other than SJS / TEN, and other skin diseases), the concentration at which the specificity is 80% or more and the Youden Index is the highest was set as the cut-off value. Numerical values of sensitivity and specificity of 80% or more with an AUC of 0.8 or more are shown in bold. Proteins with low discrimination performance with an AUC of 0.7 or less were not evaluated for cut-off values.

[0071] Although SFN showed specificity for SJS / TEN, the sensitivity of the specific diagnosis was slightly low at 60.9% (Table 7). Therefore, we focused on CD30 and TARC, which showed a low increase in SJS / TEN, and examined whether combining them could enhance the specific diagnostic performance of SJS / TEN. The specific diagnostic performance of SFN alone for SJS / TEN was an AUC of 0.79 (Table 7), but when using the ratio of SFN to TARC value (SFN / TARC ratio), the AUC value improved to 0.87. When setting the cut-off value of the SFN / TARC ratio at 5.6, it was found that the sensitivity increased to 70% or more and the specificity increased to 87% or more, enabling high-precision discrimination between SJS / TEN and other skin diseases (Table 8). JPEG0007705847000008.jpg60166From the results of the ROC curve analysis when comparing SJS / TEN and other skin diseases (including mild drug eruption, severe drug eruption other than SJS / TEN, and other skin diseases), the concentration at which the specificity is 80% or more and the Youden Index is the highest was set as the cut-off value.

[0072] 4-3-5) Comparison of positive rates in each skin disease Table 9 shows the positive rates in each disease when using the cut-off values set in Table 7. SFN showed the highest positive rate (61%) in SJS / TEN, but there were also positive cases in EM-major (45%) and autoimmune blistering diseases (47%). CD30 was the candidate protein with the highest positive rate (79%) for DIHS, and unlike TARC, there were no positive cases in autoimmune blistering diseases. DcR3 and GNLY, different from SFN and CD30, showed high positive rates in both DIHS and SJS / TEN.

[0073] For JPEG0007705847000009.jpg90170SFN, the cut-off value for specific diagnosis of SJS / TEN, and for other proteins, the cut-off values for specific diagnosis of DIHS (Table 7) were used to calculate the respective positive rates. Positive rates of 50% or more are shown in bold. HC: Healthy, Mild: Mild drug eruption, DIHS: Acute stage of DIHS, SJS / TEN: Acute stage of SJS / TEN, AGEP: Acute generalized exanthematous pustulosis, EM-major: Severe form of erythema multiforme, Atopic: Atopic dermatitis, Psoriasis: Psoriasis, Autoimmune: Autoimmune blistering disease, Infection: Infectious skin disease.

[0074] Table 10 shows the results of comparing the positive rates when combining each biomarker. In each combination, when both or either one shows a value equal to or higher than the cut-off value, it is judged as positive. When combining CD30 and TARC, the positive rate for DIHS improved to 90% or more, and the combinations of CD30 and GNLY, and CD30 and IL-1Ra also showed an improvement to 85% or more. Also, the positive rate for SJS / TEN was highest when using the SFN / TARC ratio (positive rate 74%). These results indicated the usefulness of using these biomarker candidate proteins in combination.

[0075] For JPEG0007705847000010.jpg78170SFN, the cut-off value for specific diagnosis of SJS / TEN and the cut-off values for specific diagnosis of DIHS for other proteins were used for evaluation. Positive rates of 60% or more are shown in bold. HC: Healthy, Mild: Mild drug eruption, DIHS: Acute stage of DIHS, SJS / TEN: Acute stage of SJS / TEN, AGEP: Acute generalized exanthematous pustulosis, EM-major: Severe form of erythema multiforme, Atopic: Atopic dermatitis, Psoriasis: Psoriasis, Autoimmune: Autoimmune blistering disease, Infection: Infectious skin disease. All publications, patents, and patent applications cited in this specification are hereby incorporated by reference in their entirety.

Industrial Applicability

[0076] The present invention can be used for in vitro diagnostic agents, clinical examinations, etc.

Claims

**Claim 1** A method for examining severe drug eruption, comprising measuring the expression of at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a sample derived from a subject. **Claim 2** The method according to claim 1, further comprising measuring the expression of at least one protein selected from the group consisting of granulysin (GNLY) and CC motif chemokine 17 (TARC / CCL17) in a sample derived from a subject. **Claim 3** The method according to claim 1 or 2, wherein the protein whose expression is measured is at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), and the measured value aids in the diagnosis of the severity of severe drug eruption. **Claim 4** The method according to claim 1 or 2, wherein the protein whose expression is measured is at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), and the measured value aids in the diagnosis of the disease trend of severe drug eruption. **Claim 5** The method according to claim 1 or 2, wherein the protein whose expression is measured is stratifin, and the measured value aids in the diagnosis of drug hypersensitivity syndrome and / or Stevens-Johnson syndrome / toxic epidermal necrolysis. **Claim 6** The method according to claim 1 or 2, wherein the protein whose expression is measured is TNF receptor superfamily member 8 (CD30 / TNFRSF8), and the measured value aids in the diagnosis of drug hypersensitivity syndrome and / or Stevens-Johnson syndrome / toxic epidermal necrolysis. **Claim 7** The protein for which expression is measured is at least one combination of proteins selected from the group consisting of a combination of stratifin and C-C motif chemokine 17 (TARC / CCL17) and a combination of stratifin and TNF receptor superfamily member 8 (CD30 / TNFRSF8), and the method according to claim 1 or 2, wherein the measured value aids in the specific diagnosis of drug hypersensitivity syndrome or Stevens-Johnson syndrome / toxic epidermal necrolysis.

8. The protein for which expression is measured is at least one combination of proteins selected from the group consisting of a combination of TNF receptor superfamily member 8 (CD30 / TNFRSF8) and C-C motif chemokine 17 (TARC / CCL17), a combination of TNF receptor superfamily member 8 (CD30 / TNFRSF8) and granulysin (GNLY), and a combination of TNF receptor superfamily member 8 (CD30 / TNFRSF8) and interleukin-1 receptor antagonist (IL-1Ra), and the method according to claim 1, wherein the measured value aids in the specific diagnosis of drug hypersensitivity syndrome.

9. A kit for the examination of severe drug eruption, comprising a reagent capable of measuring the expression in a sample derived from a subject for at least one protein selected from the group consisting of stratifin, TNF receptor superfamily member 8 (CD30 / TNFRSF8), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B).

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