Protein diagnostic biomarkers for severe drug eruptions

Abstract

Development of biomarkers for the diagnosis of the condition, severity, disease type of severe drug eruptions such as drug hypersensitivity syndrome and Stevens-Johnson syndrome / toxic epidermal necrolysis. A method for detecting severe drug eruptions, comprising the steps of: measuring the expression of at least one protein selected from the group consisting of a stratified protein, 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] This invention relates to a method for assisting in the diagnosis of the onset and severity of severe drug eruptions. Background Technology

[0002] Severe drug eruptions such as Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and drug hypersensitivity syndrome (DIHS) can leave serious sequelae and sometimes even lead to death. Therefore, early diagnosis and prompt initiation of appropriate treatment are crucial. Consequently, there is a need for biomarkers capable of early diagnosis of DIHS and SJS / TEN.

[0003] To date, although sufficient validation is needed for blood protein markers for severe drug eruptions, TARC (chemokine CCL17), granlysin (GNLY), FAS-L, interleukin-6 (IL-6), IP-10 (chemokine CXCL10), and combinations thereof have been proposed (Non-Patent Literature 1, 2, 3).

[0004] Stratifin (SFN), also known as 14-3-3σ, is a protein with a molecular weight of 27,000. Increased expression of SFN in lung tissue may be associated with carcinogenesis (Non-Patent Document 4). In addition, its gene expression in skin tissue can be induced by ultraviolet radiation exposure, and a technology has been developed to use this as an indicator to detect the degree of skin damage (Patent Document 1).

[0005] CD30 (TNFRSF8) is a member of the TNF receptor superfamily and a 120 kDa type I transmembrane protein. It is known to be expressed in activated lymphocytes in healthy individuals and strongly expressed in tumor cells of Hodgkin's lymphoma in hematologic malignancies. Therefore, the detection of CD30 based on histoimmunological methods for the diagnosis of Hodgkin's lymphoma has attracted much attention. Recently, it has also been shown to be expressed in undifferentiated large cell lymphoma and non-hematologic malignancies, and anti-CD30 antibody therapies have been developed (Non-Patent Literature 5).

[0006] However, it has long been unknown that SFN and soluble CD30 present in the blood can detect SJS / TEN and DIHS, and their effectiveness is unclear. In addition, the correlation between interleukin-1 receptor antagonists (IL-1Ra) and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) and the pathogenesis of severe drug eruptions is also unknown.

[0007] Existing technical documents

[0008] Non-patent literature

[0009] Non-patent document 1: Komatsu-Fujii T et al. The thymus and activation-regulatedchemokine (TARC) level in serum at an early stage of a drug eruption is aprognostic biomarker of severity of systemic inflammation, Allergology Int. 67, 90-95, 2018

[0010] Non-patent document 2: Abe R, et al. Rapid immunochromatographic test for serumgranulysin is useful for the prediction of Stevens-Johnson syndrome and toxicepidermal necrolysis. J Am Acad Dermatol. 2011, 65(1):65-8.

[0011] Non-patent document 3: Shiohara T, et al. Monitoring the acute response in severehypersensitivity reactions to drugs. Curr Opin Allergy Clin Immunol. 2015, 15(4): 294-9.

[0012] Non-patent literature 4: Shiba-Ishii A, Noguchi M. Aberrant stratifin overexpression is regulated by tumor-associated CpG demethylation in lung adenocarcinoma. AmJ Pathol. 2012Apr; 180(4):1653-62.

[0013] Non-Patent Literature 5: Junichi Tamaru. CD30. Modern Media, Vol. 61, No. 11, 2015, Advances in Clinical Testing, 323, 11-14

[0014] Patent documents

[0015] Patent Document 1: Japanese Patent Application Publication No. 2012-39970 Summary of the Invention

[0016] The problem the invention aims to solve

[0017] The purpose of this invention is to provide: a method for assisting in the high-precision diagnosis of SJK / TEN syndrome and drug hypersensitivity syndrome (DIHS), a method for objectively evaluating their therapeutic efficacy, and a method for assisting in the differentiation between curable mild drug eruption cases that will not become severe and cases that will become severe.

[0018] Solution for solving the problem

[0019] Based on plasma proteomic analysis of patients with severe drug eruptions using the SOMAscan assay, we explored biomarkers that are considered useful for diagnosing the severity or disease progression of SJK / TEN syndrome and drug hypersensitivity syndrome (DIHS). Based on the measurements of 1310 proteins, we explored proteins that showed significant changes during the acute phase of these severe drug eruptions. In addition to the previously reported TARC (chemokine CCL17) and granzyme lysin (GNLY), we identified four proteins: 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). Validation was performed using ELISA. The results showed that CD30 was the protein with the highest diagnostic ability for DIHS (identification of acute and convalescent phases) and the ability to differentiate severe cases (differentiation from mild drug eruptions), demonstrating superior results compared to previously reported markers such as granzyme and TARC. Furthermore, SFN showed the best biomarker performance in the diagnosis of SJS / TEN and in differentiating severe cases. In addition, combining these proteins can improve the positive rate of DIHS and SJS / TEN, or effectively differentiate SJS / TEN from DIHS with different treatment strategies. In particular, the use of the SFN / TARC ratio is useful for the specific diagnosis of SJS / TEN (differential diagnosis from DIHS).

[0020] This invention is based on these insights. The main points of this invention are as follows.

[0021] (1) A method for detecting severe drug eruption, comprising the following steps: determining the expression of at least one protein selected from the group consisting of layered protein, 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.

[0022] (2) The method according to (1) further includes the following step: determining the expression of at least one protein selected from the group consisting of particulate lysin (GNLY) and CC motif chemokine 17 (TARC / CCL17) in a sample from the subject.

[0023] (3) The method according to (1) or (2), wherein the expressed protein is selected from at least one protein in the group consisting of layered protein, 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 assists in the severe diagnosis of severe drug eruption.

[0024] (4) The method according to (1) or (2), wherein the expressed protein is selected from at least one protein in the group consisting of layered protein, 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 assists in the diagnosis of severe drug eruption.

[0025] (5) The method according to (1) or (2), wherein the expressed protein is a layered protein and the measured value is used to aid in the diagnosis of drug hypersensitivity syndrome and / or Schwarz-Jones syndrome / toxic epidermal necrolysis.

[0026] (6) The method according to (1) or (2), wherein the expressed protein is TNF receptor superfamily member 8 (CD30 / TNFRSF8), and the measured value is used to aid in the diagnosis of drug hypersensitivity syndrome and / or Schwarz-Jones syndrome / toxic epidermal necrolysis.

[0027] (7) According to the method of (1) or (2), wherein the expressed protein is selected from at least one group of proteins selected from the combination of layer protein and CC motif chemokine 17 (TARC / CCL17) and the combination of layer protein and TNF receptor superfamily member 8 (CD30 / TNFRSF8), and the measured value is used to assist in the specific diagnosis of drug hypersensitivity syndrome or Sch-Johnson syndrome / toxic epidermal necrolysis.

[0028] (8) A diagnostic method for severe drug eruption, comprising the following steps:

[0029] a3. The sample is obtained from the test subject;

[0030] b3. The expression of at least one protein selected from the group consisting of layered proteins, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) was determined in a subject-derived sample; and

[0031] c3. Based on the measured value of b3, determine the severity of drug eruption.

[0032] (9) A diagnostic method for severe drug eruption, comprising the following steps:

[0033] a2. The sample is obtained from the test subject;

[0034] b2. For at least one protein selected from the group consisting of layered proteins, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), the expression of the protein in a subject-derived sample was determined; and

[0035] c2. Based on the measured value of b2, determine the condition of severe drug eruption.

[0036] (10) A diagnostic method for severe drug eruption, comprising the following steps:

[0037] a4. The sample is obtained from the test subject;

[0038] b4. The expression of at least one protein selected from the group consisting of layered proteins, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) was determined in a subject-derived sample; and

[0039] c4. Based on the measured value of b4, determine the disease type of severe drug eruption.

[0040] (11) A method for diagnosing and treating severe drug eruption, comprising the following steps:

[0041] a3. The sample is obtained from the test subject;

[0042] b3. For at least one protein selected from the group consisting of layered protein, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), the expression of the protein in a sample from the subject was determined.

[0043] c3. Based on the measured values ​​of b3, determine the severity of the drug eruption; and

[0044] d3. Discontinue the use of any suspected drug that the subject is deemed to have a high probability of developing or has already developed a severe drug rash, and begin appropriate treatment based on the severity of the rash.

[0045] (12) A method for diagnosing and treating severe drug eruption, comprising the following steps:

[0046] a2. The sample is obtained from the test subject;

[0047] b2. For at least one protein selected from the group consisting of layered protein, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), the expression of the protein in a sample from the subject was determined.

[0048] c2. Based on the measured values ​​of b2, assess the severity of severe drug eruption; and

[0049] d2. Discontinue the suspected medication being taken by the subject who is deemed to be in the acute phase of a severe drug eruption, and begin appropriate treatment.

[0050] (13) A method for diagnosing and treating severe drug eruption, comprising the following steps:

[0051] a4. The sample is obtained from the test subject;

[0052] b4. For at least one protein selected from the group consisting of layered protein, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), the expression of the protein in a sample from the subject was determined.

[0053] c4. Based on the measured values ​​of b4, determine the disease type of severe drug eruption; and

[0054] d4. Discontinue the suspected medication being taken by the subject who has been diagnosed with severe drug eruption, and begin appropriate treatment based on the type of severe drug eruption.

[0055] (14) A kit for detecting severe drug eruption, comprising reagents that can measure the expression of at least one protein selected from the group consisting of layered proteins, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) in a subject-derived sample.

[0056] The effects of the invention

[0057] For severe drug eruptions, initiating treatment as early as possible is crucial. The proteins discovered by the inventors have become novel blood markers for differentiating between DIHS, SJS / TEN, and mild cases (severity discrimination markers), and further useful markers for diagnosing whether DIHS and SJS / TEN patients have entered the recovery phase (disease diagnosis markers). Additionally, they serve as useful markers for determining whether a patient's disease type is DIHS or SJS / TEN (disease type determination markers). In particular, the layering protein and CD30 exhibit higher accuracy than existing marker candidates such as TARC and granzyme, and by combining them with existing markers such as TARC and granzyme, early detection and specific diagnosis of DIHS and SJS / TEN patients can be achieved.

[0058] This specification includes the contents set forth in the specification and / or drawings of Japanese Patent Application No. 2020-73955, which forms the basis of the priority claim of this application. Attached Figure Description

[0059] Figure 1 This refers to the results of SOMAscan assays conducted during the exploration phase and the validation results based on ELISA. Disc refers to the Discovery-set (exploratory test subjects), and Vali refers to the Validation-set (validation test subjects). HC: Healthy, Mild: Mild drug eruption, DIHS: Acute phase of DIHS, SJS / TEN: Acute phase of SJS / TEN, Other: Acute phase of other severe drug eruptions (acute generalized exanthematous pustulosis and severe erythema multiforme), All-R: Recovery phase.

[0060] Figure 2This shows the distribution of blood concentrations of various biomarkers in healthy individuals, those with mild drug eruptions, those with severe drug eruptions, and various skin diseases. Results from the combined group are shown. HC: Healthy; Mild: Mild drug eruption; DIHS: Acute phase of DIHS; SJS / TEN: Acute phase of SJS / TEN; AGEP: Acute phase of acute generalized exanthematous pustulosis; EM-major: Acute phase of severe erythema multiforme; All-R: Recovery group; Atopic dermatitis: Atopic dermatitis; Psoriasis: Psoriasis; Autoimmune: Autoimmune herpes; Infection: Infectious skin diseases. Detailed Implementation

[0061] The embodiments of the present invention will be described in detail below.

[0062] The present invention provides a method for detecting severe drug eruption, comprising the following steps: determining the expression of at least one protein selected from the group consisting of layered protein, 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.

[0063] Drug eruption is a rash that occurs in response to a drug and is a side effect. It usually develops 1-2 weeks after starting medication and usually subsides upon discontinuing the causative drug, making diagnosis relatively easy. However, severe drug eruptions, which are serious in nature, often do not improve simply by discontinuing the causative drug. Without proper treatment, they can be life-threatening, making early diagnosis crucial.

[0064] Sjögren's syndrome (SJS) and toxic epidermal necrosis (TEN), representative examples of severe drug eruptions, have high mortality rates and are difficult-to-treat diseases with sequelae such as residual blindness and respiratory complications. SJS and TEN are considered to be in the same spectrum of diseases. While TEN symptoms are similar to SJS, they are more severe, and TEN often develops from SJS. In SJS, extensive mucosal lesions are observed at the mucocutaneous junction of the lips, conjunctiva, and vulva, accompanied by fever. These lesions are characterized by erosions / vesicles based on necrotic epidermal lesions accompanied by generalized erythema. The affected area is less than 10% of the body surface area. On the other hand, TEN is characterized by extensive erythema, vesicles covering more than 10% of the body surface, significant necrotic epidermal lesions such as epidermal peeling / erosion, accompanied by high fever and mucosal rashes. It is considered the most severe skin lesion caused by drugs. The incidence rates of SJS and TEN are 1–6 per 1 million people per year and 0.4–1.3 per 1 million people per year, respectively.

[0065] Drug-induced hypersensitivity syndrome (DIHS) is a severe form of drug eruption, similar to SJS / TEN. It is accompanied by high fever above 38 degrees Celsius, and systemic erythematous papules and erythema multiforme are observed, which may progress to erythroderma. Mucosal rashes are usually absent or mild, but erosion of the oral mucosa is sometimes observed. Systemic lymphadenopathy, organ dysfunction primarily due to liver dysfunction, and peripheral leukocyte abnormalities (leukocytosis, eosinophilia, and the presence of atypical lymphocytes) may be observed. A characteristic feature is the reactivation of human herpesvirus (HHV) 6 during the process; if lymphadenopathy or HHV-6 reactivation cannot be confirmed, atypical DIHS is diagnosed. Unlike typical drug eruptions, symptoms often appear more than two weeks after administration of the causative drug, and may persist even after discontinuation of the causative drug, generally requiring more than one month for relief.

[0066] For these severe drug eruptions, early diagnosis and prompt initiation of appropriate treatment are considered important. However, the initial symptoms are similar to those of mild drug eruptions, making it difficult to identify or predict patients who will develop severe illness early on. Furthermore, when treating patients who develop severe illness, it becomes crucial to know the patient's disease type early and to appropriately manage the condition (symptom activity).

[0067] Previously, it was proposed that TARC (chemokine CCL17), known as a biomarker for assessing the severity of atopic dermatitis, could be used as a blood protein marker for the validation of DIHS, or for the detection of SJS / TEN, granzyme, Fas ligand, etc. could be used as blood protein markers for the detection of severe drug eruptions.

[0068] Layer protein, also known as 14-3-3σ, is a 248-amino acid protein encoded by the SFN gene. It is known to be expressed in the epithelium of the esophagus and skin, and its expression is elevated in head and neck cancer, lung cancer, and cervical cancer. In the epidermis of the skin, layer protein expression increases due to ultraviolet radiation and is p53-dependent, acting on dermal fibroblasts and participating in wound healing and fibrosis control. Its UniProt code is P31947.

[0069] TNF receptor superfamily member 8 (CD30 / TNFRSF8) is a type I transmembrane protein of the TNF receptor family, encoded by the TNFRSF8 gene, consisting of 593 amino acids. In healthy adults, it is only observed to be expressed in active T and B cells, but in Hodgkin lymphoma, it is strongly expressed in mononuclear Hodgkin cells, multinucleated Reed-Stedberg cells, and undifferentiated large cell lymphoma. CD30 activates NFκB through phosphorylation of IκB, and is associated with cytokine secretion, inflammation regulation, cell survival, and proliferation. In clinical testing, it has been used as a marker for adult T-cell leukemia, Hodgkin lymphoma, and Ki-1 lymphoma in cellular immunoassays. It has been reported that soluble CD30 is cleaved and released through CD30L binding, and its expression increases under oxidative stress. UniProt ID: P28098.

[0070] Interleukin-1 receptor antagonist (IL-1Ra) is a 159-amino acid extracellular secretory protein encoded by the IL1RN gene. It binds to the receptor IL-1R1, inhibiting the activation of interleukin-1α and -1β. High expression has been observed in bone marrow and immune system cells, as well as in cells of the digestive tract. It is known to participate in regulating skin aging in skin tissue. Its UniProt number is P18510.

[0071] TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), formally registered as TNF receptor superfamily member 6B, is a 245-amino acid protein encoded by the gene TNFRSF6. It is known to be a decoy receptor belonging to the TNF receptor family. It inhibits Fas-induced apoptosis signaling by binding to Fas ligands as a decoy receptor. Elevated serum DcR3 levels are known in patients with rheumatoid arthritis. Its Uniprot number is O95407.

[0072] Layering proteins, CD30, IL-1Ra, and DcR3 / TNFRSF6B are novel and useful biomarkers for severe drug eruptions.

[0073] In the method of this invention, preferably, the expressed protein is a layered protein, and the measured value aids in the diagnosis of drug hypersensitivity syndrome (DIHS) and / or Schwann-Jones syndrome / toxic epidermal necrolysis (SJS / TEN). The diagnosis includes disease diagnosis, severity diagnosis, and disease-type-specific diagnosis.

[0074] Alternatively, in the method of the present invention, it is preferable to measure the expressed protein as CD30, and the measured value assists in the diagnosis of DIHS and / or SJS / TEN. The diagnosis includes disease diagnosis, severity diagnosis, and disease-type-specific diagnosis.

[0075] Furthermore, in the method of the present invention, the expression of proteins known as globulin (GNLY), TARC, and other blood protein markers for severe drug eruptions can be measured in samples derived from the test subject. By combining these novel markers with each other or with known markers, the positive rate for severe drug eruptions can be improved, and the specificity can be enhanced.

[0076] In this invention, the subject is a mammal suspected of developing a severe drug eruption, but any mammal considered to be at risk of developing the disease can be used. Humans are typical. Samples derived from the subject can include cells, tissues, body fluids, etc., specifically the subject's blood (e.g., whole blood, serum, plasma, extracorporeal plasma exchange fluid, etc.), bronchoalveolar lavage fluid, etc. Whole blood, serum, or plasma obtained from routine blood tests (clinical tests) can be used as blood samples.

[0077] In the method of the present invention, the determination of expression in a sample derived from the test subject involves measuring the amount of the aforementioned protein or its fragments present in the sample. Known methods can be used without particular limitation as the means of determination. Determination at the protein level is preferred, but determination at the nucleic acid level is also possible.

[0078] To determine the expression of the aforementioned proteins at the protein level, antibodies that specifically recognize these proteins can be used. The antibodies can be either monoclonal or polyclonal antibodies. These antibodies can be manufactured using known methods, or commercially available ones can be used. Typical methods include immunoassays such as ELISA and immunochromatography. Immunoassays do not require special equipment or techniques and can easily and rapidly detect and quantify target proteins; therefore, they are preferred for the determination of the aforementioned proteins in this invention. Antibodies for the aforementioned proteins are known and commercially available. Furthermore, as mentioned above, the amino acid sequences of the aforementioned proteins and the base sequences encoding them are also known; therefore, specific antibodies against each protein can be prepared by fabricating a conventional hybridoma.

[0079] Immunoassays are well known in the art. Depending on the reaction method, there are sandwich assays, competitive assays, agglutination assays, immunoblotting, etc. Additionally, based on labeling, there are enzyme immunoassays, radioimmunoassays, fluorescence immunoassays, luminescent immunoassays, etc. In this invention, any type of immunoassay capable of quantitative detection can be used. There are no particular limitations; for example, sandwich assays such as sandwich ELISA are preferred. In a sandwich assay, an antibody bound to a 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, etc. The detection antibody is preferably an antibody that binds to the target protein at a different site than the immobilized antibody. Both the immobilized antibody and the detection antibody can be polyclonal or monoclonal antibodies, and antigen-binding fragments of the antibody can also be used. The target protein bound to the immobilized antibody is reacted with the detection antibody, washed, and the amount of bound detection antibody is measured by a signal from a substance labeled on the antibody. For example, when using an antibody labeled with alkaline phosphatase as a detection antibody, the substrate of the enzyme is added to the reaction system, and the amount of color, fluorescence, or luminescence produced by the enzyme reaction is measured using an instrument. For standard samples containing a known concentration of the target protein, an immunoassay is performed, and a standard curve showing the relationship between the signal and concentration of the labeled substance is pre-prepared. The same procedure is performed for samples with an unknown concentration of the target protein. By substituting the obtained signal measurement value into the standard curve, the target protein in the sample can be quantified.

[0080] To determine the expression of the aforementioned proteins at the nucleic acid level, nucleic acid probes capable of specifically hybridizing to the mRNA of these proteins can be used (in the case of RNA blotting). Alternatively, at least one pair of nucleic acid primers capable of specifically amplifying cDNA synthesized using the mRNA of the aforementioned proteins as a template can be used (in the case of RT-PCR). Nucleic acid probes and primers can be designed based on the genetic information of the aforementioned proteins (as described above). For nucleic acid probes, a length of approximately 15–1500 bases is generally suitable. Nucleic acid probes can be labeled with radioactive elements, fluorescent dyes, enzymes, etc. For nucleic acid primers, a length of approximately 15–30 bases is generally suitable. Nucleic acid primers can also be labeled with radioactive elements, fluorescent dyes, enzymes, etc.

[0081] The expressed protein or gene can be one or more. By referring to data on the expression of multiple genes and proteins, a more accurate evaluation can be achieved. To simultaneously detect the expression of multiple genes and proteins, detection methods such as DNA arrays (probes immobilized on a substrate) (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, DECEMBER 2002, 951-960), protein chips (antibodies immobilized on a substrate) (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, SEPTEMBER 2002, 683-695), and Luminex (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, JUNE 2002, 447-456) can be used.

[0082] Examples of combinations of proteins or genes expressed as described in Table 10 of the embodiments described below, and ratios (CD30+TARC, CD30+SFN, CD30+GNLY, CD30+IL-1Ra, SFN / TARC ratio, SFN / CD30 ratio), are available, but are not limited to these.

[0083] For at least one protein selected from the group consisting of layered protein, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), the expression level in the sample from the subject can be measured. Based on the expression level, the severity of severe drug eruption, the severity of the disease, and the identification of the disease type can be performed.

[0084] Therefore, the method of the present invention can assist in the diagnosis of severe drug eruption (diagnosis of the condition, diagnosis of its severity, and diagnosis of the specific type of disease).

[0085] As an example of the present invention, the diagnosis of severe drug eruption can be based on the following criteria: The expression of at least one of the aforementioned proteins in plasma collected from the subject is measured. If a value higher than a pre-set cutoff value or a baseline value is obtained, the subject is evaluated as having developed severe drug eruption. This pre-set cutoff value can be appropriately set by those skilled in the art. For example, the baseline value can be the 95% confidence interval of the quantitative value of a healthy individual who has not developed severe drug eruption, or the cutoff value can be set based on an ROC curve. Alternatively, if at least one of the aforementioned proteins shows an increasing trend compared to past measurements, severe drug eruption is suspected. For the cutoff values ​​of the candidate biomarkers shown in Tables 5 to 8 of the embodiments described later, ROC curve analysis of the acute phase of DIHS or SJS / TEN and the target groups (total recovery group, mild group, or various skin disease groups) is performed, and the values ​​are set with reference to the Youden Index (sensitivity + specificity - 100).

[0086] The method of this invention can be used for the severe diagnosis of severe drug eruptions. In this specification, "severe diagnosis" refers to the differentiation between severe drug eruptions such as SJS / TEN and DIHS and mild drug eruptions such as disseminated maculopapular rashes and erythema multiforme. For at least one protein selected from the group consisting of layering protein, CD30, IL-1Ra, and DcR3, a high expression level in the sample from the subject indicates a high probability of future or existing severe drug eruptions.

[0087] As cutoff values ​​for differentiating, for example, DIHS, SJS / TEN and mild drug eruptions, they can be set as follows: layering protein: 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.

[0088] This invention provides a diagnostic method for severe drug eruption, comprising the following steps:

[0089] a3. The sample is obtained from the test subject;

[0090] b3. The expression of at least one protein selected from the group consisting of layered proteins, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) was determined in a subject-derived sample; and

[0091] c3. Based on the measured value of b3, determine the severity of drug eruption.

[0092] Furthermore, the method of this invention can be used for the diagnosis of severe drug eruptions. In this specification, "diagnosis" refers to the identification of the acute and recovery phases of severe drug eruptions such as SJS / TEN and DIHS. A high expression level of at least one protein selected from the group consisting of layering protein, CD30, IL-1Ra, and DcR3 in a sample from the subject indicates a high probability of being in the acute phase of a severe drug eruption.

[0093] As cutoff values ​​for diagnosing conditions such as DIHS and SJS / TEN, they can be set as follows: layered protein: 1-3 ng / mL (preferably 1.0 ng / mL), DcR3: 0.3-2 ng / mL (preferably 1.1 ng / mL), IL-1Ra: 500-1500 pg / mL (preferably 930 pg / mL), CD30: 50-150 ng / mL (100 ng / mL), etc.

[0094] This invention provides a diagnostic method for severe drug eruption, comprising the following steps:

[0095] a2. The sample is obtained from the test subject;

[0096] b2. For at least one protein selected from the group consisting of layered proteins, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), the expression of the protein in a subject-derived sample was determined; and

[0097] c2. Based on the measured value of b2, determine the condition of severe drug eruption.

[0098] SFN exhibits high detection performance, particularly for SJS / TEN, and CD30 shows high detection performance, especially for DIHS. DcR3 and IL-1Ra are effective at detecting both.

[0099] Furthermore, the method of the present invention can be used for the specific diagnosis of severe drug eruptions. In this specification, "specific diagnosis" refers to the identification of the disease type of severe drug eruptions. Severe drug eruptions such as SJS / TEN and DIHS sometimes lead to serious sequelae or death; therefore, its specific diagnostic value is of great clinical significance. For example, a high expression level of layering protein in a sample from the subject indicates a high probability of SJS / TEN. Similarly, a high expression level of CD30 in a sample from the subject indicates a high probability of DIHS. To improve the detection rate of severe drug eruptions, biomarkers can be combined. The combination of layering protein and TARC can improve the identification performance of SJS / TEN. The SFN / TARC ratio can be used to identify SJS / TEN. Additionally, the detection performance of DIHS can be improved by combining CD30 with TARC, CD30 with layering protein, CD30 with granzyme-like protein (GNLY), and CD30 with IL-1Ra.

[0100] For example, the cutoff values ​​for specific diagnosis of SJS / TEN can be set as follows: layered protein: 3-7 ng / mL (preferably 4.3 ng / mL); and the cutoff values ​​for specific diagnosis of DIHS can be set as follows: 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). To improve the detection performance of DIHS and SJS / TEN, these cutoff values ​​can be combined for each marker. Furthermore, even when combined with previously reported TARC (for example, cutoff value: 5500 pg / mL) and GNLY (for example, cutoff value: 10.7 ng / mL), the detection performance of DIHS and SJS / TEN can be improved. The cutoff value for the SFN / TARC ratio to improve the differential detection performance of SJS / TEN can be set to 4-7 (preferably 5.6).

[0101] This invention provides a diagnostic method for severe drug eruption, comprising the following steps:

[0102] a4. The sample is obtained from the test subject;

[0103] b4. The expression of at least one protein selected from the group consisting of layered proteins, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B) was determined in a subject-derived sample; and

[0104] c4. Based on the measured value of b4, determine the disease type of severe drug eruption.

[0105] The method of this invention can be used not only for diagnosing severe drug eruptions but also for prognostic assessment and confirmation of treatment efficacy. For example, for at least one protein selected from the group consisting of layering protein, CD30, IL-1Ra, and DcR3, its expression is measured once or multiple times at different times in samples from subjects deemed highly likely to be in the acute phase of severe drug eruption. When the expression level decreases to near a cutoff value or baseline value, recovery from severe drug eruption is determined through treatment; if the aforementioned level is high or does not decrease, recovery from severe drug eruption is determined to be incomplete or unsuccessful. The cutoff value used to determine recovery through treatment is valid for disease diagnosis.

[0106] If the likelihood of a severe drug eruption occurring or already occurring in the subject is deemed high, the suspected drug should be discontinued, and appropriate treatment should be initiated based on the patient's condition and the specific symptoms associated with the disease type. As a pharmacological therapy for DIHS, systemic administration of steroids (converted to prednisolone, starting at 0.5–1 mg / kg / day and gradually decreasing appropriately) is effective; however, dosage reduction should be considered to address the recurrence of symptoms due to HHV-6 reactivation. In cases of SJS / TEN, systemic administration of different doses of steroids is recommended based on the severity of the illness (converted to prednisolone: ​​0.5–1 mg / kg / day for moderate cases, 1–2 mg / kg / day for severe cases, and methylprednisolone for the most severe cases, starting at 1 g / day for 3 days and gradually decreasing as appropriate according to symptoms). In addition, high-dose intravenous immunoglobulin (IVIG) therapy (400 mg / kg / day for 5 consecutive days, in principle only one course of treatment), plasma exchange therapy, ocular surface inflammation treatment for eye lesions (betamethasone or dexamethasone eye drops (about 4 times a day)), inhibition of eyelid adhesions to preserve the ocular surface epithelium, and prevention of ocular surface infections, etc.

[0107] In the United States, severe drug eruptions are also a serious side effect. Regarding SJS / TEN, the Genetic and Rare Diseases Information Center (GARD), funded by the National Institutes of Health (NIH), publishes information on symptoms, causes, treatments, and prognoses (https: / / rarediseases.info.nih.gov / diseases / 7700 / Stevens-Johnson-syndrometoxic-epidermal-necrolysis). Treatment options include maintenance therapy and pharmacological therapy, in addition to requiring hospitalization and discontinuation of the administered medication. Maintenance therapy typically treats skin symptoms similarly to that for severe burns, including wound care, pain management, fluid and electrolyte balance, nutritional support, temperature management, and monitoring or treatment of secondary infections. Additionally, eye symptoms may be treated with saline rinsing. Pharmacological therapies include systemic corticosteroids, intravenous immunoglobulin (IVIG), cyclosporine, plasma exchange, and anti-tumor necrosis factor (TNF) monoclonal antibodies, but these have not been adequately studied through randomized trials. Regarding ophthalmic medication treatment, examples include: multiple daily lubrication with preservative-free eye drops or ointments; and ophthalmic medication treatment including topical corticosteroids and broad-spectrum antibiotics. Additionally, in a 2018 survey of 22 university hospitals, among 377 patients with SJS / TEN, maintenance therapy alone accounted for 29.3%, steroids alone for 30.0%, IVIG therapy for 24.9%, and a combination of steroids and IVIG therapy for 14.3% (Michaeletti RG et al., J InvestDermatol., 2018; 138:2315-2321).

[0108] Regarding DIHS (also known as Drug Reaction with Eosinophilia and Systemic Symptoms, DRESS, or drug reaction accompanied by eosinophilia and systemic symptoms), while GARD describes the symptoms and clinical studies, it does not describe the treatment (https: / / rarediseases.info.nih.gov / diseases / 13629 / drug-reaction-with-eosinophilia-and-systemic-symptoms). In an analysis of electronic medical information from Partners HealthCare System in Boston, out of 3,162,562 patients, 69 received DRESS and 51 received corticosteroids. Of these, 35 received topical steroids, 36 received oral steroids, and 26 received intravenous steroids (Wolfson AR et al., J Allergy Clin Immunol Pract., 2019; 7:633-640).

[0109] This invention provides a method for diagnosing and treating severe drug eruptions, comprising the following steps:

[0110] a3. The sample is obtained from the test subject;

[0111] b3. For at least one protein selected from the group consisting of layered protein, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), the expression of the protein in a sample from the subject was determined.

[0112] c3. Based on the measured values ​​of b3, determine the severity of the drug eruption; and

[0113] d3. Discontinue the use of any suspected drug that the subject is deemed to have a high probability of developing or has already developed a severe drug rash, and begin appropriate treatment based on the severity of the rash.

[0114] In addition, the present invention provides a method for diagnosing and treating severe drug eruptions, comprising the following steps:

[0115] a2. The sample is obtained from the test subject;

[0116] b2. For at least one protein selected from the group consisting of layered protein, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), the expression of the protein in a sample from the subject was determined.

[0117] c2. Based on the measured values ​​of b2, assess the severity of severe drug eruption; and

[0118] d2. Discontinue the suspected medication being taken by the subject who is deemed to be in the acute phase of a severe drug eruption, and begin appropriate treatment.

[0119] Furthermore, the present invention provides a method for diagnosing and treating severe drug eruptions, comprising the following steps:

[0120] a4. The sample is obtained from the test subject;

[0121] b4. For at least one protein selected from the group consisting of layered protein, TNF receptor superfamily member 8 (CD30 / TNFRSF8), interleukin-1 receptor antagonist (IL-1Ra), and TNF receptor superfamily member 6B (DcR3 / TNFRSF6B), the expression of the protein in a sample from the subject was determined.

[0122] c4. Based on the measured values ​​of b4, determine the disease type of severe drug eruption; and

[0123] d4. Discontinue the suspected medication being taken by the subject who has been diagnosed with severe drug eruption, and begin appropriate treatment based on the type of severe drug eruption.

[0124] The present invention also provides a kit for detecting severe drug eruption, comprising reagents that can measure the expression of at least one protein selected from the group consisting of layered proteins, 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 test subject.

[0125] As an example, the kit of the present invention contains an antibody as a reagent capable of specifically recognizing the aforementioned protein. The antibody can be immobilized on a microtiter plate, magnetic beads, cellulose membrane, or substrate. The kit may further include equipment for collecting samples from the subject, an anticoagulant, a set of reagents for detecting the aforementioned protein, and an instruction manual. In addition to the method of using the kit, the instruction manual may also pre-describe the evaluation and / or identification criteria for the acute phase of severe drug eruptions.

[0126] As another example, the kit of the present invention includes nucleic acid probes capable of specifically hybridizing with the mRNA of the aforementioned proteins as reagents. The nucleic acid probes may also be immobilized on a substrate. The kit may further include apparatus for collecting biological samples, anticoagulants, reagents for extracting RNA from samples from the subject, reagents for detecting RNA, and instructions for use. In addition to the method of using the kit, the instructions for use may also pre-describe the evaluation and / or identification criteria for the acute phase of severe drug eruptions.

[0127] As another example, the kit of the present invention includes, as a reagent, at least one pair of nucleic acid primers capable of specifically amplifying cDNA synthesized using the mRNA of the aforementioned protein as a template. The kit may further include equipment for collecting samples from the subject, an anticoagulant, reagents for extracting RNA from the sample from the subject, reagents for detecting RNA, and instructions for use. In addition to the method of using the kit, the instructions for use may also pre-describe the evaluation and / or identification criteria for the acute phase of severe drug eruptions.

[0128] The kit of the present invention may also include standard proteins, buffer solution, substrate (in the case of enzyme labeling of antibodies), reaction stop solution, washing solution, reaction vessel, etc.

[0129] In addition to diagnosing severe drug eruptions, the kit of this invention can also diagnose SJS caused by infections such as mycoplasma, and can be used as a drug for diagnosing diseases.

[0130] Example

[0131] The present invention will be further described in detail below through embodiments.

[0132] [Example 1]

[0133] (1) Specimen

[0134] For samples from patients with severe drug eruptions used for analysis, collection was conducted with the permission of the research ethics committees of designated hospitals, the National Institute of Food and Drug Control, the Kihara Foundation, Astellas Pharma Inc., and Daiichi Sankyo. Blood samples were collected from patients suspected of having mild drug eruptions (erythema multiforme, disseminated maculopapular rash, eczematous rash), and patients with related skin diseases at Yokohama City University, Shimane University, Shimada Municipal Hospital, Iwata Municipal General Hospital, Nara Medical University, and Niigata University. Additionally, blood samples were collected from patients with skin diseases other than drug eruptions (atopic dermatitis, psoriasis, autoimmune herpes, and infectious skin diseases (excluding measles, rubella, and chickenpox)), and their plasma and serum were cryopreserved at -80°C. Samples from healthy individuals were collected by NOYES Corporation.

[0135] Details of the test samples are shown in Table 1. Seventy-five case samples and 12 healthy samples collected up to November 2017 were used for exploration and validation of the ELISA kit based on SOMAscan analysis (exploration group, Table 1). Next, as a separate group from the exploration group, test samples collected after December 2017 (163 case samples and 38 healthy samples not used in the exploration) were used for biomarker validation (validation set). Furthermore, a combined group incorporating relevant diseases was used to evaluate the biomarker performance, combining the exploration and validation groups.

[0136] Table 1. Details of the specimens used for analysis

[0137]

[0138] (2) Exploration of biomarker candidates

[0139] Biomarker exploration was conducted for two clinically significant severe drug eruption types, SJS / TEN and DIHS, using proteomic analysis with the SOMAscan system from SomaLogic. The SOMAscan system is a assay that detects 1,310 proteins using aptamers (artificial ligands based on single-stranded nucleic acids). Plasma samples from the exploration group were sent to SomaLogic, USA, in a frozen state for SOMAscan analysis.

[0140] Based on SOMAscan measurements of 1,310 proteins, we explored (1) candidate markers for DIHS and SJS / TEN that could differentiate the disease (comparison between acute and recovery phases of severe illness) and (2) candidate markers for severe illness-related markers that could be distinguished from the mild case group (comparison between acute and mild case groups). After logarithmically transforming the measured values ​​(fluorescence signal intensity) of each probe, the patients were divided into four groups: Mild (n=29), DIHS acute phase group (DHIS, n=14), SJS / TEN acute phase group (n=9), All-R (n=19), and healthy individuals (HC, n=12). In any group of DIHS or SJS / TEN acute phase, proteins with an effect size Hedge's g value and / or fold change (FC) ranking within the top 10, and exhibiting an AUC value greater than 0.85 in focused ROC curve analysis, were found to contain TARC and GNLY, known as previously reported markers of severe drug eruptions, as well as previously unknown markers of severe drug eruptions such as layering proteins, CD30, DcR3, and IL-1Ra. Table 2 shows the ELISA assay systems used in these biomarker candidates and subsequent validation experiments.

[0141] Table 2. Biomarker candidate proteins and ELISA assay system for validation

[0142] Biomarker candidates Uniprot ID ELISA kit matrix Layered protein (SFN) P31947 Homemade ELISA serum CD30 / TNFRSF8 P28908 Invitrogen serum DcR3 / TNFRSF6B O95407 R&D company Duoset plasma IL-1Ra P18510 R&D company Quantikine plasma Particulate Lysate (GNLY) [already reported] P22749 Homemade ELISA serum TARC / CCL17 [already reported] Q92583 Sysmex, HISCL TARC serum

[0143] (3) Comparison of measurement results

[0144] Figure 1 This section presents the variation patterns of SOMAscan and ELISA measurements for six candidate biomarkers. In SOMAscan analysis, all six proteins showed significant increases in all disease types of severe drug eruptions compared to the healthy group and the mild drug eruption group, decreasing to near-healthy levels in the recovery group. Similar results were obtained in ELISA; the concentration ranges in the healthy group, DIHS, the magnitude of increase in the acute phase of SJS / TEN cases, and the overall concentration range in the recovery group were similar in both the exploration and validation groups. Figure 1 ).

[0145] (4) Usefulness as a diagnostic tool for the disease and for determining its severity

[0146] 4-1) Evaluation based on the exploration group

[0147] Table 3 compares the disease discrimination performance (differentiation between severe acute and recovery phases) and severe disease discrimination ability (differentiation between severe drug rash group and mild group) of each candidate protein observed in the exploration group for DIHS and SJS / TEN with the AUC values ​​calculated by ROC curve analysis. SFN, DcR3, and IL-1Ra are predicted to have high discrimination performance for both DIHS and SJS / TEN, with SFN showing particularly high detection performance for SJS / TEN. In contrast, CD30 shows high detection performance for DIHS, with its disease discrimination ability (AUC 0.93) and severe disease diagnostic performance (AUC 0.87) being higher than those of the previously reported TARC (AUC 0.89 and 0.82).

[0148] Table 3. Comparison of AUC values ​​of various biomarker candidates with disease severity and severity assessment capabilities for DIHS and SJS / TEN (exploratory group)

[0149]

[0150] Values ​​greater than 0.85 are indicated in bold.

[0151] 4-2) Validation based on validation groups

[0152] Table 4 shows the ROC analysis results for the validation group. Similar to the exploration group, in the validation group, SFN showed high discriminative ability, particularly for SJS / TEN, CD30 showed high discriminative ability for DIHS, and DcR3 and IL-1Ra showed discriminative ability for both. When comparing the disease discrimination and severity diagnosis ability for DIHS, DcR3 (AUC 0.99 and 0.86) and CD30 (AUC 0.94 and 0.89) were higher than the previously reported TARC (AUC 0.91 and 0.78) and GNLY (AUC 0.86 and 0.81). Regarding the disease discrimination ability for SJS / TEN, SFN (AUC 0.92) showed the highest value. The ability of SFN to differentiate severe SJS / TEN observed in the exploration group (AUC 0.91, Table 3) was slightly reduced in the validation group (AUC 0.79). In the combined group of the exploration and validation groups, SFN had the highest AUC (AUC 0.84), followed by IL-1Ra (AUC 0.80), which was also higher than the previously reported GNLY (AUC 0.76) (Table 4).

[0153] Table 4. Comparison of AUC values ​​of each biomarker candidate with the ability to detect the severity and severity of DIHS and SJS / TEN (validation group).

[0154]

[0155] Values ​​greater than 0.85 are indicated in bold.

[0156] 4-3) Evaluation results based on joint groups

[0157] 4-3-1) Disease specificity

[0158] Figure 2 This study compared the distribution of measurements of various candidate proteins in a range of skin diseases, including drug eruptions, using data from a combined cohort. For any candidate protein, strong increases were observed in DIHS and / or SJS / TEN, with SFN showing a characteristic increase in SJS / TEN, while only moderate increases were observed in other disease types, including DIHS, EM-major, or autoimmune herpes. On the other hand, CD30 and TARC showed similar DIHS-specific increases, mild increases in a portion of EM-major, but almost no increases in autoimmune herpes. DcR3 and GNLY, unlike SFN and CD30, showed an increasing tendency in both DIHS and SJS / TEN.

[0159] 4-3-2) Differentiation of DIHS and SJS / TEN conditions

[0160] Table 5 presents the data analysis results of the combined group, showing the disease discrimination performance of each candidate protein for DIHS and SJS / TEN. Cutoff values ​​for the acute phase and total recovery phase (All-R) were set to identify DIHS and SJS / TEN, and sensitivity and specificity were compared. In DIHS disease discrimination, DcR3 (AUC 0.96) and CD30 (AUC 0.94) showed excellent performance. With a cutoff value of 1.1 ng / mL for DcR3 and 100 ng / mL for CD30, the sensitivity was above 80% and the specificity above 90%, both capable of distinguishing between the acute and total recovery phases of DIHS. Both DcR3 and SFN exhibit excellent disease discrimination performance in SJS / TEN (both AUCs are above 0.9). With a cutoff value of 0.55 ng / mL, DcR3 has a sensitivity of over 90% and a specificity of over 85%, which can distinguish between the acute phase and the total recovery phase of SJS / TEN. With a cutoff value of 1.0 ng / mL, SFN has both a sensitivity and specificity of over 80%, which can also distinguish between the acute phase and the total recovery phase of SJS / TEN.

[0161] Table 5. Cutoff values ​​and performance for differentiating DIHS and SJS / TEN (combined group).

[0162]

[0163] Based on the ROC curve analysis results comparing the acute phase and total recovery phase (All-R) of DIHS and SJS / TEN, the cutoff value was set at a specificity of ≥80% and the highest Youden Index. Values ​​with an AUC of ≥0.85 are marked in bold. Proteins with low recognition performance (AUC below 0.7) were not evaluated for the cutoff value.

[0164] 4-3-3) Differentiation of severe cases in DIHS and SJS / TEN

[0165] Table 6 presents the analysis results of the combined group data, showing the performance of each candidate protein in differentiating between DIHS and SJS / TEN for severe cases. Cutoff values ​​were set for identifying the acute and mild (mild) phases of DIHS and SJS / TEN, and sensitivity and specificity were compared. In the severe case differentiation of DIHS, CD30 (AUC 0.88) was the most effective, exceeding the previously reported values ​​for GNLY (AUC 0.85) and TARC (AUC 0.79). With a CD30 cutoff value of 82 ng / mL, both sensitivity and specificity were above 80% (Table 6). On the other hand, in the severe case differentiation of SJS / TEN, SFN was the most effective (AUC 0.84), with a cutoff value of 3.7 ng / mL, achieving a sensitivity of approximately 70% and a specificity of approximately 90%, effectively differentiating between the acute and mild phases of SJS / TEN.

[0166] Table 6. Cutoff values ​​and performance for determining the severity of DIHS and SJS / TEN (combination group).

[0167]

[0168] Based on the ROC curve analysis results comparing the acute and mild cases of DIHS and SJS / TEN, the cutoff value was set at a specificity of ≥80% and the highest Youden Index. Sensitivity and specificity values ​​of ≥80% with an AUC of ≥0.8 are marked in bold. Proteins with low recognition performance (AUC below 0.7) were not evaluated for the cutoff value.

[0169] 4-3-4) Specific diagnosis of disease type in severe drug eruption

[0170] Severe drug eruptions, particularly SJS / TEN and DIHS, are serious disease types. Due to their different treatment strategies, early diagnosis of SJS / TEN versus DIHS is crucial. Therefore, this study compares the performance of various proteins in detecting DIHS from various skin diseases (DIHS-specific diagnostic performance) and their performance in specifically detecting SJS / TEN (SJS / TEN-specific diagnostic performance).

[0171] Table 7 presents the results of comparing sensitivity and specificity of DIHS with other skin diseases (including mild drug eruptions, severe drug eruptions other than DIHS, and other skin diseases) and SJS / TEN with other skin diseases (including mild drug eruptions, severe drug eruptions other than DIHS, and other skin diseases), setting suitable cutoff values ​​for specific diagnosis of DIHS and SJS / TEN. For DIHS, CD30 showed the best specific diagnostic performance (AUC 0.87), with a cutoff value of 124 ng / mL, both sensitivity and specificity were approximately 80%. For SJS / TEN, SFN showed the highest specific diagnostic performance (AUC 0.79), with a cutoff value of 4.3 ng / mL, sensitivity was 60.9%, and specificity was 81.8%.

[0172] Table 7. Cutoff values ​​and performance comparison for specific diagnosis of DIHS and SJS / TEN

[0173]

[0174] Based on the ROC curve analysis results comparing DIHS with other skin diseases (including mild drug eruptions, severe drug eruptions other than DIHS, and other skin diseases), and SJS / TEN with other skin diseases (including mild drug eruptions, severe drug eruptions other than SJS / TEN, and other skin diseases), the cutoff value was set at a specificity of ≥80% and the highest Youden Index. Sensitivity and specificity values ​​of ≥80% with an AUC of ≥0.8 are marked in bold. Proteins with low recognition performance (AUC below 0.7) were not evaluated for cutoff values.

[0175] While SFN showed specificity for SJS / TEN, its sensitivity for specific diagnosis was slightly low at 60.9% (Table 7). Therefore, focusing on the low levels of CD30 and TARC in SJS / TEN, the study investigated whether combining SFN with these components improved the specific diagnostic performance of SJS / TEN. The specific diagnostic performance of SFN alone for SJS / TEN was AUC 0.79 (Table 7), which increased to 0.87 when using the ratio to TARC (SFN / TARC ratio). With an SFN / TARC ratio of 5.6 as the cutoff value, sensitivity increased to over 70% and specificity to over 87%, indicating the ability to identify SJS / TEN with other skin diseases with high accuracy (Table 8).

[0176] Table 8. Improvement in the diagnostic performance of SJS / TEN based on the combination of SFN and TARC

[0177]

[0178] Based on the ROC curve analysis results when comparing SJS / TEN with other skin diseases (including mild drug eruptions, severe drug eruptions other than SJS / TEN, and other skin diseases), the concentration with a specificity of over 80% and the highest Youden Index was set as the cutoff value.

[0179] 4-3-5) Comparison of positive rates in various skin diseases

[0180] Table 9 shows the positivity rates for each disease when the cutoff values ​​set in Table 7 were used. SFN showed the highest positivity rate (61%) in SJS / TEN, but positive cases were also found in EM-major (45%) and autoimmune herpes (47%). CD30 was the candidate protein with the highest positivity rate (79%) against DIHS, and unlike TARC, no positive cases were found in autoimmune herpes. DcR3 and GNLY, unlike SFN and CD30, showed high positivity rates in both DIHS and SJS / TEN.

[0181] Table 9. Comparison of positive rates of various biomarker candidates in various skin diseases.

[0182]

[0183] For SFN, the cutoff value for specific diagnosis of SJS / TEN was used; for other proteins, the cutoff value for specific diagnosis of DIHS was used (Table 7). Positive rates of 50% or higher are indicated in bold. HC: Healthy; Mild: Mild drug eruption; DIHS: Acute phase of DIHS; SJS / TEN: Acute phase of SJS / TEN; AGEP: Acute generalized exanthematous pustulosis; EM-major: Severe erythema multiforme; Atopic dermatitis: Atopic dermatitis; Psoriasis: Psoriasis; Autoimmune: Autoimmune herpes; Infection: Infectious skin disease.

[0184] Table 10 compares the positive rates when various biomarkers are combined. In each combination, a positive result is defined as either or both of the biomarkers exceeding a cutoff value. For DIHS, the combination of CD30 and TARC showed a positive rate exceeding 90%, while the combinations of CD30 and GNLY, and CD30 and IL-1Ra, showed increases exceeding 85%. Furthermore, the highest positive rate (74%) was observed for SJS / TEN when the SFN / TARC ratio was used. These results demonstrate the usefulness of combining these biomarker candidate proteins.

[0185] Table 10. Improvement in the positive rate of severe drug eruption based on combinations of candidate biomarkers.

[0186]

[0187] For SFN, the cutoff value used for specific diagnosis of SJS / TEN was used for evaluation; for other proteins, the cutoff value used for specific diagnosis of DIHS was used for evaluation. A positive rate of ≥60% is indicated in bold. HC: Healthy; Mild: Mild drug eruption; DIHS: Acute phase of DIHS; SJS / TEN: Acute phase of SJS / TEN; AGEP: Acute generalized exanthematous pustulosis; EM-major: Severe erythema multiforme; Atopic dermatitis: Atopic dermatitis; Psoriasis: Psoriasis; Autoimmune: Autoimmune herpes; Infection: Infectious skin disease.

[0188] All publications, patents and patent applications referenced in this specification are incorporated herein by reference directly.

[0189] Industrial availability

[0190] This invention can be used in in vitro diagnostic drugs, clinical testing, etc.

Claims

1. Use of a reagent for detecting a layered protein, or a combination of a layered protein and TNF receptor superfamily member 8, CD30 / TNFRSF8, in the manufacture of a medicament for detecting a severe drug eruption by a method comprising the steps of: determining expression of a layered protein, or a combination of a layered protein and TNF receptor superfamily member 8, CD30 / TNFRSF8, in a test sample derived from a subject; wherein the severe drug eruption is drug hypersensitivity syndrome and / or Stevens-Johnson syndrome / toxic epidermal necrolysis, the test sample is selected from the group consisting of plasma and serum.

2. The use according to claim 1, wherein the method further comprises the step of: determining expression of at least one protein selected from the group consisting of granulysin, GNLY, and CC chemokine ligand 17, TARC / CCL17, in a test sample derived from a subject.

3. Use according to claim 1 or 2, wherein, The protein whose expression is determined is a layered protein, or a combination of a layered protein and TNF receptor superfamily member 8, CD30 / TNFRSF8, and the determined value assists in the diagnosis of severity of a severe drug eruption; wherein the diagnosis of severity is a discrimination between a severe drug eruption and a mild drug eruption.

4. The use according to claim 1 or 2, wherein, The protein whose expression is determined is a layered protein, or a combination of a layered protein and TNF receptor superfamily member 8, CD30 / TNFRSF8, and the determined value assists in the diagnosis of the condition of a severe drug eruption, wherein the diagnosis of the condition is a discrimination between an acute phase and a recovery phase of a severe drug eruption.

5. The use according to claim 1 or 2, wherein, The protein whose expression is determined is a layered protein, and the determined value assists in the diagnosis of drug hypersensitivity syndrome and / or Stevens-Johnson syndrome / toxic epidermal necrolysis.

6. The use according to claim 1 or 2, wherein, The protein whose expression is determined is TNF receptor superfamily member 8, CD30 / TNFRSF8, and the determined value assists in the diagnosis of drug hypersensitivity syndrome and / or Stevens-Johnson syndrome / toxic epidermal necrolysis.

7. The use according to claim 1 or 2, wherein, The protein whose expression is determined is a combination of at least one protein selected from the group consisting of a combination of a layered protein and CC chemokine ligand 17, TARC / CCL17, and a combination of a layered protein and TNF receptor superfamily member 8, CD30 / TNFRSF8, and the determined value assists in the specific diagnosis of drug hypersensitivity syndrome or Stevens-Johnson syndrome / toxic epidermal necrolysis.

8. Use of a reagent for detecting a layered protein, or a combination of a layered protein and TNF receptor superfamily member 8, CD30 / TNFRSF8, in the manufacture of a medicament for diagnosing severity of a severe drug eruption by a method comprising the steps of: a3. obtaining a test sample from a subject; b3. determining expression of a layered protein, or a combination of a layered protein and TNF receptor superfamily member 8, CD30 / TNFRSF8, in a test sample derived from a subject; and c3. determining the severity of a drug eruption based on the determined value of b3; wherein the severe drug eruption is drug hypersensitivity syndrome and / or Stevens-Johnson syndrome / toxic epidermal necrolysis, the diagnosis of severity is a discrimination between a severe drug eruption and a mild drug eruption, the test sample is selected from the group consisting of plasma and serum, The judgment of the severity of drug eruptions refers to the discrimination of severe drug eruptions from mild drug eruptions.

9. Use of a reagent for detecting a layered protein, or a combination of a layered protein and TNF receptor superfamily member 8, CD30 / TNFRSF8, in the manufacture of a medicament for the diagnosis of the condition of severe drug eruptions by a method comprising the steps of: a2. obtaining a test sample from a subject; b2. measuring the expression of a layered protein, or a combination of a layered protein and TNF receptor superfamily member 8, CD30 / TNFRSF8, in the test sample derived from the subject; and c2. judging the condition of severe drug eruptions based on the measured value of b2; wherein the severe drug eruptions are drug hypersensitivity syndrome and / or Stevens-Johnson syndrome / toxic epidermal necrolysis, the test sample is selected from the group consisting of plasma and serum, the judgment of the condition of severe drug eruptions refers to the identification of the acute phase and the recovery phase of severe drug eruptions.

10. Use of a reagent for detecting a layered protein, or a combination of a layered protein and TNF receptor superfamily member 8, CD30 / TNFRSF8, in the manufacture of a medicament for the specific diagnosis of severe drug eruptions by a method comprising the steps of: a4. obtaining a test sample from a subject; b4. measuring the expression of a layered protein, or a combination of a layered protein and TNF receptor superfamily member 8, CD30 / TNFRSF8, in the test sample derived from the subject; and c4. judging the disease type of severe drug eruptions based on the measured value of b4; wherein, the severe drug eruptions are drug hypersensitivity syndrome and / or Stevens-Johnson syndrome / toxic epidermal necrolysis, the test sample is selected from the group consisting of plasma and serum.

11. Use of a reagent for detecting a layered protein, or a combination of a layered protein and TNF receptor superfamily member 8, CD30 / TNFRSF8, in the manufacture of a kit for detecting severe drug eruptions, the reagent being capable of measuring the expression of a layered protein, or a combination of a layered protein and TNF receptor superfamily member 8, CD30 / TNFRSF8, in a test sample derived from a subject; wherein the severe drug eruptions are drug hypersensitivity syndrome and / or Stevens-Johnson syndrome / toxic epidermal necrolysis, the test sample is selected from the group consisting of plasma and serum.

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