A novel biomarker for evaluating the severity and prognosis of dermatomyositis / antisynthetase syndrome-related interstitial lung disease and its application

Abstract

The present invention discloses a novel biomarker for evaluating the severity and prognosis of dermatomyositis / anti-synthetase syndrome-related interstitial lung disease and its application. The present invention finds through research that the content expression of sTREM-1 in the sera of all patients with dermatomyositis / anti-synthetase-related interstitial lung disease, especially those with rapidly progressive interstitial lung disease, is significantly increased. To a certain extent, the content of serum sTREM-1 in patients with dermatomyositis / anti-synthetase-related interstitial lung disease reflects the expression of the membrane receptor TREM-1 in lung tissues. The proposal of the present invention enables early warning of the risk of RP-ILD in patients with dermatomyositis / anti-synthetase complicated with interstitial lung disease at the initial stage of the disease, providing an effective technical means for improving the prognosis of patients with dermatomyositis / anti-synthetase syndrome complicated with ILD, especially RP-ILD patients.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and relates to a novel biomarker for evaluating the severity and prognosis of dermatomyositis / antisynthetase syndrome-related interstitial lung disease and its application. Background Art

[0002] Idiopathic inflammatory myopathy (IIM) is an autoimmune disease with muscle weakness as the typical clinical manifestation, which can be accompanied by multiple organ involvement such as skin, joints, and lungs. According to clinical, histopathological, and serological characteristics, IIM can be divided into five subtypes: dermatomyositis (DM), antisynthetase syndrome (ASS), immune-mediated necrotizing myopathy (IMNM), inclusion body myositis (IBM), and polymyositis (PM). Interstitial lung disease (ILD) is the main manifestation of IIM in the lungs, and among them, patients with DM and ASS are most likely to be combined with ILD. Some of these patients show progressive worsening of dyspnea accompanied by imaging deterioration within 3 months, which is called rapidly progressive interstitial lung disease (RPILD). Once patients are combined with RPILD, the prognosis is extremely poor, and the mortality rate within half a year is as high as 41%. However, the current mechanism of IIM-related interstitial lung disease is not clear, and there is a lack of effective biomarkers for early identification of the rapid progression of IIM-related interstitial lung disease.

[0003] Triggering receptor expressed on myeloid cells-1 (TREM-1) is an immunoglobulin superfamily molecule mainly expressed on the surface of myeloid cells such as neutrophils, monocytes, and macrophages. It participates in various inflammatory responses by stimulating the production of pro-inflammatory chemokines and cytokines. In addition to existing in the form of membrane-expressed receptors, TREM-1 also has a soluble form (soluble form of TREM-1, sTREM-1) that can be detected in serum and bronchoalveolar lavage fluid. Currently, there are two hypotheses about the source of sTREM-1. One is that sTREM-1 is formed by the variant translation of the TREM-1 mRNA spliceosome, and the other is that the membrane-expressed TREM-1 form is formed after being shed from the membrane. Regardless of the source of sTREM-1, multiple studies have shown that TREM-1 (including sTREM-1) can be used as a detection index for the disease activity of various autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease. However, it is still unclear whether it participates in the pathogenesis of dermatomyositis-associated interstitial lung disease and whether it is related to the disease severity and prognosis. Summary of the Invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0005] The present invention provides the application of a reagent for detecting the TREM-1 biomarker in a sample in the preparation of a product for diagnosing whether a subject has dermatomyositis / antisynthetase syndrome-related interstitial lung disease.

[0006] Furthermore, the interstitial lung disease includes rapidly progressive interstitial lung disease or non-rapidly progressive interstitial lung disease.

[0007] Furthermore, the disease type of the subject diagnosed by the reagent for the TREM-1 biomarker in the sample is the severity of dermatomyositis / antisynthetase syndrome interstitial lung disease.

[0008] Furthermore, the disease type of the subject diagnosed by the reagent for the TREM-1 biomarker in the sample is whether dermatomyositis / antisynthetase syndrome is complicated with interstitial lung disease.

[0009] Furthermore, the disease type of the subject diagnosed by the reagent for the TREM-1 biomarker in the sample is whether dermatomyositis / antisynthetase syndrome is complicated with rapidly progressive interstitial lung disease.

[0010] Furthermore, the TREM-1 is soluble TREM-1.

[0011] As used herein, the term "TREM-1" refers to triggering receptor expressed on myeloid cells-1. The term "sTREM-1" refers to soluble form of triggering receptor expressed on myeloid cells-1.

[0012] As used herein, the term "dermatomyositis" is synonymous with "DM" and refers to dermatomyositis. The term "antisynthetase syndrome" is synonymous with "ASS" and refers to antisynthetase syndrome. The term "interstitial lung disease" is synonymous with "ILD" and refers to interstitial lung disease. The term "rapidly progressive interstitial lung disease" is synonymous with "RP ILD" and refers to rapidly progressive interstitial lung disease. The term "non-rapidly progressive interstitial lung disease" is synonymous with "non-RP ILD".

[0013] Further, the sample includes bone marrow, blood, blood cells, serum, plasma, ascites, tissue or fine needle biopsy sample, cell-containing body fluid, free-floating nucleic acid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluid, skin swab, oral swab, nasal swab, rinse fluid, lavage fluid, aspirate, scrape, bone marrow sample, tissue biopsy sample, surgical sample, secretion and / or excrement.

[0014] Further, the sample includes blood, blood cells, serum, plasma, bronchoalveolar lavage fluid.

[0015] Further, the subject includes a human or non-human mammal.

[0016] Further, the subject is a human.

[0017] In some specific embodiments, suitable mammals falling within the scope of the present invention include vertebrates, specifically including but not limited to any member of the subphylum Chordata, including primates, and including monkey species, rodents, lagomorphs, cattle, sheep, goats, pigs, horses, dogs, cats, avians (e.g., chickens, turkeys, ducks, geese, companion birds (such as canaries, budgerigars, etc.)), marine mammals, reptiles, and fish.

[0018] Further, the product includes a reagent, kit, chip, or test strip.

[0019] Furthermore, the product determines whether a subject has dermatomyositis / anti-synthetase syndrome-related interstitial lung disease or the risk of having dermatomyositis / anti-synthetase syndrome-related interstitial lung disease by measuring the level of TREM-1 in a sample.

[0020] Furthermore, the reagent includes specific primers, probes, antisense oligonucleotides, aptamers or antibodies for detecting biomarkers.

[0021] Furthermore, the reagent includes reagents used in the following methods: TaqMan probe method, sequencing method, chip method, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), restriction fragment length polymorphism (RFLP) method, single-strand conformation polymorphism (SSCP) method, allele-specific PCR, SNaPshot method, SNPlex method, denaturing high-performance liquid chromatography (DHPLC) method, denaturing gradient gel electrophoresis (DGGE) method.

[0022] Furthermore, the kit includes a reagent for extracting a biomarker and a reagent for quantitatively detecting the biomarker.

[0023] Furthermore, the quantitative detection reagent includes: primers for specifically amplifying a biomarker, probes for specifically recognizing a biomarker, and binding agents for specifically binding to a biomarker protein.

[0024] Furthermore, the binding agent includes an antibody for specifically binding to a biomarker protein, a functional fragment of an antibody, and a conjugated antibody.

[0025] Furthermore, the binding agent is an antibody for specifically binding to a biomarker protein.

[0026] As used herein, the term "probe" refers to a material for detecting a probe that binds to a biomarker. The label can be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Specifically, the label can be any one selected from the group consisting of quantum dots, HRP, and fluorescent proteins. In one embodiment, the label can be GFP (green fluorescent protein), BFP (blue fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), or HRP (horseradish peroxidase), but is not limited thereto.

[0027] The present invention provides the use of a reagent for detecting the TREM-1 biomarker in a sample in the preparation of a product for predicting the prognosis of patients with dermatomyositis / anti-synthetase syndrome-related interstitial lung disease.

[0028] Furthermore, the interstitial lung disease includes rapidly progressive interstitial lung disease or non-rapidly progressive interstitial lung disease.

[0029] Furthermore, the TREM-1 is soluble TREM-1.

[0030] Further, the sample includes bone marrow, blood, blood cells, serum, plasma, ascites, tissue or fine needle biopsy sample, cell-containing body fluid, free-floating nucleic acid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluid, skin swab, oral swab, nasal swab, rinse fluid, lavage fluid, aspirate, scrape, bone marrow sample, tissue biopsy sample, surgical sample, secretion and / or excrement.

[0031] Further, the sample includes blood, blood cells, serum, plasma, bronchoalveolar lavage fluid.

[0032] Further, the subject includes a human or non-human mammal.

[0033] Further, the subject is a human.

[0034] Further, the product determines the prognosis risk of a patient with dermatomyositis / anti-synthetase syndrome-related interstitial lung disease by measuring the level of TREM-1 in the sample.

[0035] Further, the prognosis risk of a patient with dermatomyositis / anti-synthetase syndrome-related interstitial lung disease is determined by the level of TREM-1. When the expression level of TREM-1 is greater than or equal to 373.8 ng / ml, the prognosis risk of the patient is high; when the expression level of TREM-1 is less than 373.8 ng / ml, the prognosis risk of the patient is low.

[0036] Further, the reagent includes a specific primer, probe, antisense oligonucleotide, aptamer or antibody for detecting a biomarker.

[0037] Further, the reagent includes the reagents used in the following methods: TaqMan probe method, sequencing method, chip method, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry detection, restriction fragment length polymorphism method, single-strand conformation polymorphism method, allele-specific PCR, SNaPshot method, SNPlex method, denaturing high performance liquid chromatography method, denaturing gradient gel electrophoresis method.

[0038] The present invention provides a product for diagnosing dermatomyositis / anti-synthetase syndrome-related interstitial lung disease or predicting the prognosis of a patient with dermatomyositis / anti-synthetase syndrome-related interstitial lung disease, and the product includes a reagent for detecting the gene or protein expression level of the TREM-1 biomarker in a sample.

[0039] Further, the product includes a reagent, a kit, a chip or a test strip.

[0040] Further, the reagent includes a reagent for detecting mRNA and a reagent for detecting protein.

[0041] In some embodiments, the reagents for detecting mRNA include, but are not limited to: reaction reagents for visualizing the amplicons corresponding to the primers, such as reagents for visualizing amplicons by agarose gel electrophoresis, enzyme-linked gel method, chemiluminescence method, in situ hybridization method, fluorescence detection method, etc.; RNA extraction reagents; reverse transcription reagents; cDNA amplification reagents; standards used for preparing standard curves; positive control samples. In some embodiments, the reagents for detecting proteins include, but are not limited to: blocking solution, antibody diluent, washing buffer, chromogenic termination solution.

[0042] The present invention provides a computer system for diagnosing dermatomyositis / anti-synthetase syndrome-related interstitial lung disease or predicting the prognosis of patients with dermatomyositis / anti-synthetase syndrome-related interstitial lung disease, comprising:

[0043] An input unit, the computer obtains data to be processed, and the data to be processed includes the detection result of the expression level of TREM-1 in a sample;

[0044] An evaluation unit, inputs the data to be processed into a diagnostic model or a prognostic risk assessment model to obtain a computer diagnostic result or a computer prognostic risk assessment result of the data to be processed. The diagnostic model or the prognostic risk assessment model is a pre-trained model, and the computer diagnostic result or the computer prognostic risk assessment result includes the correlation between the important characteristic factor TREM-1 and the disease risk and prognostic risk of dermatomyositis / anti-synthetase syndrome-related interstitial lung disease;

[0045] An output unit, outputs the computer diagnostic result or the computer prognostic risk assessment result of the data to be processed.

[0046] Furthermore, the training in the pre-trained model includes:

[0047] Obtaining data of dermatomyositis / anti-synthetase syndrome-related interstitial lung disease of known samples, and the data of dermatomyositis / anti-synthetase syndrome-related interstitial lung disease includes expression result data of the biomarker TREM-1;

[0048] Dividing the data of dermatomyositis / anti-synthetase syndrome-related interstitial lung disease of the known samples into training set data and test set data;

[0049] Inputting the data into an algorithm model for training, and adjusting the model based on the test set data in the data to be processed to obtain a diagnostic model or a prognostic risk assessment model.

[0050] Further, the algorithm model includes a linear regression algorithm model, a support vector machine algorithm model, a nearest neighbor / k-nearest neighbor algorithm model, a logistic regression algorithm model, a decision tree algorithm model, a k-means algorithm model, a random forest algorithm model, a naive Bayes algorithm model, a dimensionality reduction algorithm model, and a gradient boosting algorithm model.

[0051] Further, the diagnostic model diagnoses whether a sample is dermatomyositis / anti-synthetase syndrome-related interstitial lung disease or at risk of having dermatomyositis / anti-synthetase syndrome-related interstitial lung disease by calculating the AUC value of each sample and comparing it with the optimal threshold obtained from the ROC curve generated by the prediction results of the model established based on the ensemble learning method on the test set.

[0052] Further, the prognostic risk assessment model determines by comparing the level of TREM-1 in each sample. When the expression level of TREM-1 in the sample is greater than or equal to 373.8 ng / ml, the patient has a high prognostic risk. When the expression level of TREM-1 in the sample is less than 373.8 ng / ml, the patient has a low prognostic risk.

[0053] In some embodiments, the prognostic risk assessment model is used to evaluate whether dermatomyositis / anti-synthetase syndrome-related interstitial lung disease is rapidly progressive interstitial lung disease.

[0054] The above describes specific embodiments of the present specification. Other embodiments are within the scope of the appended claims. In some cases, the actions or steps recited in the claims can be performed in a different order than in the embodiments and still achieve the desired result. Additionally, the processes depicted in the figures do not necessarily require the specific order or sequential order shown to achieve the desired result. In certain embodiments, multitasking and parallel processing are also possible or may be advantageous.

[0055] The present invention provides the application of the TREM-1 biomarker in screening drugs for treating dermatomyositis / anti-synthetase syndrome-related interstitial lung disease.

[0056] Further, the interstitial lung disease includes rapidly progressive interstitial lung disease or non-rapidly progressive interstitial lung disease.

[0057] Further, the TREM-1 is soluble TREM-1.

[0058] Further, the drug also includes a pharmaceutically acceptable excipient.

[0059] Further, the excipients include adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH regulators and / or buffers, solvents, surfactants or emulsifiers.

[0060] In certain embodiments, the drug may be formulated specifically for administration in a fixed or liquid form, including those suitable for administration by: oral administration, such as potions (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those intended for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for administration to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, such as, for example, sterile solutions or suspensions, or sustained release formulations; topical administration, such as creams, ointments, or controlled release patches or sprays for administration to the skin, lungs, or mouth; intravaginal or rectal administration, such as pessaries, creams, or foams; sublingual administration; ophthalmic administration; transdermal administration; or nasal, pulmonary administration, and administration to other mucosal surfaces.

[0061] In the context of the present invention, the term "sample" refers to a composition obtained from or derived from a subject (e.g., an individual of interest) that contains cells and / or other molecular entities to be characterized and / or identified according to, for example, physical, biochemical, chemical, and / or physiological characteristics. A "sample" can be any biological sample isolated from a subject. The term "sample" includes any biological sample that can be extracted from a subject, untreated, treated, diluted, or concentrated.

[0062] The term "comprising / including" as used in the present invention means that a composition or method of one or more named elements or steps is open-ended, meaning that the named elements or steps are required, but other elements or steps can be added within the scope of the composition and method. To avoid prolixity, it should also be understood that any composition or method described as "comprising / including" one or more named elements or steps also describes the corresponding more limited composition or method "consisting essentially of the same named elements or steps", meaning that the composition or method includes the named required elements or steps, and may also include other elements or steps that do not substantially affect the basic and novel features of the composition or method.

[0063] The term "biomarker" refers to a gene or gene product that is a target for modulating one or more target phenotypes (e.g., a target phenotype in myeloid cells). In this context, the term "biomarker" is synonymous with "target". However, in some embodiments, the term also encompasses measurable entities that have been identified as indicating a target output such as one or more diagnostic, prognostic, and / or therapeutic outputs (e.g., for modulating an inflammatory phenotype, cancer status, etc.). In other embodiments, the term also encompasses compositions that modulate a gene or gene product, including anti-gene product antibodies and antigen-binding fragments thereof. Thus, biomarkers can include, but are not limited to, nucleic acids (e.g., genomic nucleic acids and / or transcribed nucleic acids), proteins, and antibodies (and antigen-binding fragments thereof).

[0064] Advantages and beneficial effects of the present invention:

[0065] The present invention provides a novel molecular marker for predicting dermatomyositis / anti-synthetase syndrome complicated with interstitial lung disease, especially rapidly progressive interstitial lung disease. Through research, the present invention finds that the expression level of sTREM-1 in the serum of all patients with dermatomyositis / anti-synthetase-related interstitial lung disease, especially those complicated with rapidly progressive interstitial lung disease, is significantly increased. To a certain extent, the content of serum sTREM-1 in patients with dermatomyositis / anti-synthetase-related interstitial lung disease reflects the expression of membrane receptor TREM-1 in lung tissue. The present invention enables early warning of the risk of RP-ILD in patients with dermatomyositis / anti-synthetase complicated with interstitial lung disease at the initial stage of the disease, providing an effective technical means for improving the prognosis of patients with dermatomyositis / anti-synthetase syndrome complicated with ILD, especially RP-ILD patients. Brief Description of the Drawings

[0066] Figure 1 It is a graph showing the results of ELISA detection of serum sTREM-1 expression in patients with DM / ASS ILD; wherein, A is the comparison of serum sTREM-1 levels between normal control (HC) population (n = 36) and patients with DM / ASS ILD (n = 67), and B is the comparison of serum sTREM-1 levels among normal control (HC) population (n = 36), DM ILD patients positive for anti-MDA5 antibody (n = 47), ASS ILD patients (n = 11), and other types of DM ILD patients (n = 9); * represents p < 0.05, *** represents p < 0.001, and ns represents no significant difference.

[0067] Figure 2Results of detecting sTREM-1 levels in the sera of DM / ASS RP ILD patients; among them, A shows the comparison of serum sTREM-1 levels among DM / ASS patients with RP-ILD (n = 11), DM / ASS patients without RP-ILD (non-RPILD, n = 21), and healthy controls (n = 18) in the training set; B shows the comparison of serum sTREM-1 levels among DM / ASS patients with RP-ILD (n = 13), DM / ASS patients without RP-ILD (non-RPILD, n = 22), and healthy controls (n = 18) in the validation set; C shows the analysis of TREM-1 expression in the lung tissues of DM / ASS patients with RPILD, without RPILD, and normal controls (HC) by immunohistochemical staining; D shows the comparison of TREM-1 expression levels in the lung tissues of DM / ASS patients with RPILD, without RPILD, and normal controls (HC); E shows the ROC curve analysis of serum sTREM1 as a serum biomarker for differentiating DM / ASS RPILD between the training set and the test set; * represents p < 0.05, ** represents p < 0.01; *** represents p < 0.001; scale bar, 50 μm. Arrow, TREM-1 positive cells. AUC represents the area under the ROC curve.

[0068] Figure 3 Results of evaluating the prognosis of DM / ASS ILD patients by serum sTREM-1 levels; among them, A shows the comparison of serum sTREM-1 levels between surviving patients (survivors, n = 58) and deceased patients (decedents, n = 9) with DM / ASS ILD; B shows the Kaplan-Meier survival analysis of DM / ASS ILD patients grouped according to serum sTREM-1 levels; p = 0.002 by log rank test. Detailed implementation manners

[0069] The present invention is further illustrated by the following examples, which should not be construed as restrictive. The examples are merely illustrative and are not intended to limit any situation described herein in any way. The following examples do not limit the present invention in any way.

[0070] In practice, the Receiver Operating Characteristic curve (ROC curve) is typically calculated by plotting the variable values against their relative frequencies in "normal" (e.g., apparently healthy individuals without antenatal disorders or conditions) and "disease" populations (similarly, e.g., two different states such as viral and bacterial RTIs or typical and atypical pneumonias). For any particular marker, the distributions of the marker levels in subjects with and without the disease / condition may overlap. Under such conditions, the test cannot absolutely distinguish normal from disease with 100% accuracy, and the overlapping region may indicate where the test fails to distinguish normal from disease. A threshold is selected below which the test is considered "abnormal" and above which the test is considered "normal", or tests below or above the threshold indicate a particular condition. The Area Under the Curve (AUC) of the ROC curve is a measure of the probability that a perceptual measurement will allow correct identification of the condition. Even if the test results do not necessarily provide exact numbers, the ROC curve can be used. As long as the results can be ranked, an ROC curve can be created.

[0071] Those skilled in the art should be able to understand that, for any of a variety of purposes, especially in terms of providing a written description, any range provided herein also includes all possible sub-ranges and combinations of these sub-ranges, as well as the individual values that make up the range, especially integer values. Thus, it should be understood that each unit between two particular units is also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are separately disclosed, as well as their inclusion as part of the range. The recited ranges (e.g., weight percentages or carbon groups) include the individual specific values, integers, decimals, or properties within the range. It should be readily understood that any range listed can be fully described and enabled such that the same range can be divided into equal halves, thirds, fourths, fifths, or tenths. As a non-limiting example, each range described herein can be readily divided into lower third, middle third, and upper third, etc. Those skilled in the art should also be able to understand that terms such as "up to", "at least", "above", "below", "more than", "greater than", "less than", "or above", or similar terms include the recited numbers, and such terms also refer to the range that can subsequently be divided into the sub-ranges described above. Similarly, any ratio listed herein also includes sub-ratios that fall within the broader ratio.

[0072] 1. Research Subjects

[0073] Our study included 67 newly diagnosed patients with IIM complicated with ILD who were hospitalized in China-Japan Friendship Hospital from February 2016 to October 2020, including 47 DM patients positive for anti-MDA5 antibody, 11 ASS patients, and 9 DM patients of other types, including 3 DM patients positive for anti-TIF1-γ antibody, 2 DM patients positive for anti-Mi-2 antibody, 2 DM patients positive for anti-SAE1 antibody, and 2 DM patients negative for myositis-specific antibodies. In addition, 36 healthy controls matched with the patients in terms of age and gender were included. The diagnosis of DM conforms to the criteria proposed by the 239th European Neuromuscular Disease Center (ENMC), the diagnosis of ASS conforms to Connor’s criteria, the diagnosis of ILD is confirmed by high-resolution chest CT, RP-ILD is defined as the appearance of severe dyspnea symptoms within 3 months and HRCT shows rapid progression of ILD imaging changes, otherwise it is no RP-ILD.

[0074] 2. Collection of clinical data

[0075] Through the hospital's electronic medical records, the clinical manifestations and laboratory data of the patients were obtained (including basic patient information, main clinical manifestations and physical examinations, past medical history, laboratory data, etc.).

[0076] 3. Experimental methods

[0077] 3.1 All serum samples were routinely collected before treatment during the patients' hospitalization or outpatient visits and stored at -80°C. The concentration of sTREM-1 was measured using a commercial enzyme-linked immunosorbent assay kit and operated according to the manufacturer's instructions.

[0078] 3.2 Lung tissue sections of DM / ASS ILD patients were obtained by percutaneous lung biopsy or transbronchial cryobiopsy. The lung tissue of the control group was taken from normal tissues during the surgical resection of lung adenoma or hamartoma. 10% formalin was used to fix the tissues. After dehydration, the fixed tissues were embedded in paraffin (6 μm thickness) and sliced and air-dried thoroughly. After dewaxing, hydration, and antigen repair. Endogenous peroxidase was removed with 3% hydrogen peroxide, and the tissue sections were incubated with anti-TREM-1 polyclonal antibody (diluted 1:200) overnight at 4°C after blocking with goat serum. On the second day, a drop of labeled goat anti-mouse / rabbit IgG polymer was added and incubated at room temperature for 30 minutes. After washing, DAB was added for color development, counterstained with hematoxylin, and finally the tissues were dehydrated and sealed.

[0079] 4. Statistical analysis

[0080] Categorical variables were expressed as counts and percentages, normally distributed continuous variables were expressed as mean ± standard deviation (SD), and t-tests were used for comparison. Non-normally distributed data were expressed as median [interquartile range (IQR)], and Mann-Whitney U tests were used for comparison. Kruskal-Waills H tests were used for comparisons among multiple groups. P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 26.0 and GraphPad Prism 8.0.

[0081] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description of the specific embodiments. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

[0082] Example 1

[0083] Using a commercially available enzyme-linked immunosorbent assay (ELISA) kit for triggering receptor expressed on myeloid cells-1 (TREM-1) expressed by human myeloid cells (the kit contains a microplate pre-coated with a human TREM-1 specific monoclonal antibody and a horseradish peroxidase-conjugated human TREM-1 antibody), the expression of sTREM-1 in the sera of 67 DM / ASS patients and 36 age- and sex-matched healthy control (hc) subjects was preliminarily detected according to the instructions of the kit. The results showed that the sTREM-1 level in the sera of DM / ASS ILD patients was significantly higher than that in the hc group ( Figure 1 A). Further, the 67 DM / ASS ILD patients were divided into subgroups of anti-melanoma differentiation associated gene 5 (mda5) antibody-positive dermatomyositis-related ILD, ASS-related ILD, and other types of dermatomyositis-related ILD and compared with the hc group. It was found that there was no difference in the serum sTREM1 level among the three groups of DM / ASS-related interstitial lung disease patients, but it was higher than that in the hc group ( Figure 1 B). This preliminarily indicates that the sTREM-1 level is increased in DM / ASS ILD patients, and there is no obvious difference in different types of dermatomyositis complicated with ILD.

[0084] Furthermore, DM / ASS ILD patients were further divided into a rapidly progressive interstitial lung disease (RP ILD) group and a non-RP-ILD group. It was found that the sTREM-1 level in the RP ILD group was significantly higher than that in the non-RP-ILD group ( Figure 2 A - B). Through immunohistochemical staining of lung tissue, it was found that the expression of membrane receptor type TREM-1 was significantly increased in the lung tissue of the RP-ILD group ( Figure 2 C, 2D). Thus, it can be shown that the levels of both sTREM-1 and membrane receptor form TREM-1 expression are related to the severity of DM / ASS ILD. To further clarify this association, we found through binary logistic regression analysis that elevated serum sTREM-1 levels (OR: 1.049; p = 0.020), ferritin (OR: 1.093; p = 0.006), skin ulcers (OR: 12.566; p = 0.01), and fever (OR: 4.588; p = 0.033) were risk factors for the occurrence of RP-ILD (Table 1). Subsequently, an ROC curve was plotted to analyze the predictive value of serum sTREM-1 levels for the diagnosis of RPILD in DM / ASS patients. The results were as Figure 2 shown in E. The diagnostic threshold of serum sTREM-1 for the diagnosis of anti-DM / ASS-rpild in the test set was 373.8 ng / ml, the diagnostic sensitivity was 72.7%, the specificity was 76.2%, the area under the curve was 0.75, p = 0.004, and the diagnostic efficacy of the sTREM-1 threshold of 373.8 ng / ml was further verified in the validation set, with an area under the curve of 0.73 and p = 0.023.

[0085] Table 1

[0086]

[0087] Finally, 67 DM / ASS ILD patients included were followed up for a long time to further clarify the clinical value of serum sTREM-1 for predicting death. As of December 31, 2023, 9 patients died of respiratory failure. Compared with the survivors, the initial serum sTREM-1 levels in the deceased patients were significantly elevated ( Figure 3A). DM / ASS patients were divided into two groups according to whether the initial serum sTREM-1 level was less than 373.8 ng / ml. It was found that the cumulative survival rate of patients with a serum sTREM-1 level ≥ 373.8 ng / ml was significantly lower (log-rank, p = 0.002). The 2.5-year cumulative survival rate of patients with sTREM-1 ≥ 373.8 ng / ml was 70.4%, while that of patients with sTREM-1 < 373.8 ng / ml was 97.5%( Figure 3 B).

[0088] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Use of a reagent for detecting the TREM-1 biomarker in a sample in the preparation of a product for diagnosing whether a subject has dermatomyositis / antisynthetase syndrome-associated interstitial lung disease.

2. The use according to claim 1, wherein the interstitial lung disease comprises rapidly progressive interstitial lung disease or non-rapidly progressive interstitial lung disease.

3. The use according to claim 1, wherein the TREM-1 is soluble TREM-1.

4. The use according to claim 1, wherein the sample comprises a tissue or fine needle biopsy sample, a cell-containing body fluid, an aspirate, a scraping, a bone marrow sample, a tissue biopsy sample, a surgical sample, a secretion and / or an excretion.

5. The use as claimed in claim 1, wherein the sample comprises bone marrow, blood, blood cells, serum, plasma, ascites, free-floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluid, skin swab, oral swab, nasal swab, washing fluid, and lavage fluid.

6. The use according to claim 1, wherein the sample comprises blood, blood cells, serum, plasma, or bronchoalveolar lavage fluid.

7. The use according to claim 1, wherein the subject comprises a human or a non-human mammal.

8. The use according to claim 1, wherein the subject is a human.

9. The use according to claim 1, wherein the product comprises a reagent, a test kit, a chip or a test paper.

10. The use according to claim 1, wherein the product determines whether a subject has DM / AS-related interstitial lung disease or the risk of developing DM / AS-related interstitial lung disease by measuring the level of TREM-1 in a sample.

11. The use according to claim 1, wherein the reagent comprises a specific primer, a probe, an antisense oligonucleotide, an aptamer or an antibody for detecting a biomarker.

12. The use according to claim 1, wherein the reagents include reagents used in the following methods: TaqMan probe method, sequencing method, chip method, flight mass spectrometer detection, restriction fragment length polymorphism method, single-strand conformation polymorphism method, allele-specific PCR, SNaPshot method, SNPlex method, denaturing high performance liquid chromatography, and denaturing gradient gel electrophoresis.

13. Use of reagents for detecting the TREM-1 biomarker in samples for the preparation of products for predicting the prognosis of patients with dermatomyositis / antisynthetase syndrome-related interstitial lung disease.

14. The use according to claim 13, wherein the interstitial lung disease comprises rapidly progressive interstitial lung disease or non-rapidly progressive interstitial lung disease.

15. Use according to claim 13, wherein the TREM-1 is soluble TREM-1.

16. The use according to claim 13, wherein the sample comprises a tissue or fine needle biopsy sample, a cell-containing body fluid, an aspirate, a scraping, a bone marrow sample, a tissue biopsy sample, a surgical sample, a secretion and / or an excretion.

17. The use according to claim 13, wherein the sample comprises bone marrow, blood, blood cells, serum, plasma, ascites, free-floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluid, skin swab, oral swab, nasal swab, washing fluid, and lavage fluid.

18. The use according to claim 13, wherein the sample comprises blood, blood cells, serum, plasma, or bronchoalveolar lavage fluid.

19. The use according to claim 13, wherein the source of the sample comprises human or non-human mammal.

20. The use according to claim 13, wherein the sample is from a human.

21. The use according to claim 13, wherein the product determines the prognostic risk of patients with dermatomyositis / antisynthetase syndrome-related interstitial lung disease by measuring the level of TREM-1 in the sample.

22. The use according to claim 21, wherein the prognostic risk of the patient with dermatomyositis / antisynthetase syndrome-related interstitial lung disease is determined by the level of TREM-1, and when the expression level of TREM-1 is greater than or equal to 373.8 ng / ml, the patient has a high prognostic risk, and when the expression level of TREM-1 is less than 373.8 ng / ml, the patient has a low prognostic risk.

23. The use according to claim 13, wherein the reagent comprises a specific primer, a probe, an antisense oligonucleotide, an aptamer or an antibody for detecting a biomarker.

24. The use as claimed in claim 13, wherein the reagents include reagents used in the following methods: TaqMan probe method, sequencing method, chip method, flight mass spectrometer detection, restriction fragment length polymorphism method, single-strand conformation polymorphism method, allele-specific PCR, SNaPshot method, SNPlex method, denaturing high performance liquid chromatography, and denaturing gradient gel electrophoresis.

25. A product for diagnosing dermatomyositis / antisynthetase syndrome-related interstitial lung disease or predicting the prognosis of patients with dermatomyositis / antisynthetase syndrome-related interstitial lung disease, the product comprising a reagent for detecting the gene or protein expression level of the TREM-1 biomarker in a sample.

26. The product of claim 25, comprising a reagent, a test kit, a chip or a test paper.

27. A computer system for diagnosing dermatomyositis / antisynthetase syndrome-related interstitial lung disease or predicting the prognosis of a patient with dermatomyositis / antisynthetase syndrome-related interstitial lung disease, comprising: An input unit, wherein the computer obtains data to be processed, wherein the data to be processed includes a result of detecting the expression level of TREM-1 in a sample; an evaluation unit, inputting the data to be processed into a diagnostic model or a prognostic risk assessment model to obtain a computer diagnosis result or a computer prognostic risk assessment result of the data to be processed, wherein the diagnostic model or the prognostic risk assessment model is a pre-trained model, and the computer diagnosis result or the computer prognostic risk assessment result includes the correlation between the important characteristic factor TREM-1 and the risk of morbidity and prognostic risk of interstitial lung disease associated with dermatomyositis / antisynthetase syndrome; The output unit outputs the computer diagnosis result or the computer prognosis risk assessment result of the data to be processed.

28. The computer system of claim 27, wherein the training of the pre-trained model comprises: Obtaining dermatomyositis / antisynthetase syndrome-related interstitial lung disease data of known samples, wherein the dermatomyositis / antisynthetase syndrome-related interstitial lung disease data includes expression result data of the biomarker TREM-1; Dividing the dermatomyositis / antisynthetase syndrome-related interstitial lung disease data of the known samples into training set data and test set data; The data are input into an algorithm model for training, and the model is adjusted based on the test set data in the data to be processed to obtain a diagnostic model or a prognostic risk assessment model.

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