Novel marker for evaluating severity and prognosis of interstitial lung diseases related to dermatomyositis / anti-synthase syndrome and application of novel marker

Abstract

The invention discloses a novel marker for evaluating severity and prognosis of a dermatomyositis / anti-synthase syndrome related interstitial lung disease and application of the novel marker. According to the application disclosed by the invention, the content expression of sTREM-1 in serum of all patients with dermatomyositis / anti-synthase related interstitial lung diseases, especially patients with rapid progression type interstitial lung diseases, is found to be obviously increased through research. The content of the sTREM-1 in serum in patients with dermatomyositis / anti-synthase related interstitial lung diseases reflects the expression condition of the membrane receptor TREM-1 in lung tissues to a certain extent. According to the application disclosed by the invention, the risk of RP-ILD of patients suffering from dermatomyositis / anti-synthase complicated with interstitial lung diseases can be early warned at the early stage of the disease, and an effective technical means is provided for improving dermatomyositis / anti-synthase syndrome complicated with ILD, especially prognosis of the patients suffering from RP-ILD.

Description

Technical Field

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

[0002] Idiopathic inflammatory myopathy (IIM) is an autoimmune disease with muscle weakness as a typical clinical manifestation, which may be accompanied by multiple organ involvement such as skin, joints, and lungs. Based on 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 lung involvement in IIM, among which DM and ASS patients are most likely to have ILD. Some patients show progressive worsening of dyspnea and worsening of imaging within 3 months, which is called rapidly progressive interstitial lung disease (RPILD). Once patients develop RP ILD, their prognosis is extremely poor, with a mortality rate of up to 41% within six months. However, the mechanism of IIM-related interstitial lung disease is not clear at present, and there is a lack of effective markers 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 that is 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 proinflammatory chemokines and cytokines. In addition to existing in the form of membrane-expressed receptors, TREM-1 also exists in a soluble form (soluble form of TREM-1, sTREM-1) that can be detected in serum and bronchoalveolar lavage fluid. There are currently two hypotheses about the origin of sTREM-1. One is that sTREM-1 is formed by the splice variant translation of the mRNA encoding TREM-1, and the other is that the membrane-expressed TREM-1 form is formed after the release of membrane shedding. Regardless of the source of sTREM1, many studies have shown that TREM-1 (including sTREM-1) can be used as a detection indicator of the disease activity of various autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and inflammatory bowel disease. However, it remains unclear whether it is involved in the pathogenesis of dermatomyositis-related interstitial lung disease and whether it is related to 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 use of a reagent for detecting a TREM-1 biomarker in a sample in the preparation of a product for diagnosing whether a subject suffers from dermatomyositis / anti-synthetase 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 reagent for detecting the TREM-1 biomarker in the sample diagnoses the subject's disease type as the severity of dermatomyositis / anti-synthetase syndrome interstitial lung disease.

[0008] Furthermore, the reagent for detecting the TREM-1 biomarker in the sample diagnoses the subject's disease type as dermatomyositis / anti-synthetase syndrome combined with interstitial lung disease.

[0009] Furthermore, the reagent for detecting the TREM-1 biomarker in the sample diagnoses the subject's disease type as dermatomyositis / anti-synthetase syndrome combined with rapidly progressive interstitial lung disease.

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

[0011] The term "TREM-1" as used in the present invention 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] The term "dermatomyositis" used in the present invention is synonymous with "DM" and refers to dermatomyositis. The term "anti-synthetase syndrome" used in the present invention is synonymous with "ASS" and refers to antisynthetase syndrome. The term "interstitial lung disease" used in the present invention is synonymous with "ILD" and refers to intestitial lung disease. The term "rapidly progressive interstitial lung disease" used in the present invention is synonymous with "RP ILD" and refers to rapidly progressive interstitiallung disease. The term "non-rapidly progressive interstitial lung disease" used in the present invention is synonymous with "non-RP ILD".

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

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

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

[0016] Furthermore, 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, Leporidae, cattle, sheep, goats, pigs, horses, dogs, cats, birds (e.g., chickens, turkeys, ducks, geese, companion birds (such as canaries, budgies, etc.)), marine mammals, reptiles, and fish.

[0018] Furthermore, the product includes reagents, test kits, chips or test strips.

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

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

[0021] Furthermore, 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-stranded conformation polymorphism method, allele-specific PCR, SNaPshot method, SNPlex method, denaturing high performance liquid chromatography, and denaturing gradient gel electrophoresis.

[0022] Furthermore, the kit includes reagents for extracting biomarkers and reagents for quantitative detection of biomarkers.

[0023] Furthermore, the quantitative detection reagents include: primers for specifically amplifying biomarkers, probes for specifically recognizing biomarkers, and binding agents for specifically binding to biomarker proteins.

[0024] Furthermore, the binding agent includes antibodies, antibody functional fragments, and conjugated antibodies that specifically bind to biomarker proteins.

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

[0026] As used herein, the term "probe" refers to a material used to detect a probe bound to a biomarker. The label may be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Specifically, the label may be any one selected from the group consisting of quantum dots, HRP, and fluorescent proteins. In one embodiment, the label may 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 a 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 samples, cell-containing body fluids, free-floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, stool, lymph, gynecological fluid, skin swab, oral swab, nasal swab, wash fluid, lavage fluid, aspirate, scrape, bone marrow sample, tissue biopsy sample, surgical sample, secretion and / or excretion.

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

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

[0033] Furthermore, the subject is a human.

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

[0035] Furthermore, the prognostic risk of the patients with dermatomyositis / antisynthetase 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 patient's prognostic risk is high, and when the expression level of TREM-1 is less than 373.8 ng / ml, the patient's prognostic risk is low.

[0036] Furthermore, the reagents include specific primers, probes, antisense oligonucleotides, aptamers or antibodies for detecting biomarkers.

[0037] Furthermore, 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-stranded conformation polymorphism method, allele-specific PCR, SNaPshot method, SNPlex method, denaturing high performance liquid chromatography, and denaturing gradient gel electrophoresis.

[0038] The present invention provides a product 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, the product comprising a reagent for detecting the gene or protein expression level of the TREM-1 biomarker in a sample.

[0039] Furthermore, the product includes reagents, test kits, chips or test strips.

[0040] Furthermore, the reagents include reagents for detecting mRNA and reagents for detecting protein.

[0041] In some embodiments, the reagents for detecting mRNA include but are not limited to: reaction reagents for visualizing the amplicon corresponding to the primer, such as reagents for visualizing the amplicon by agarose gel electrophoresis, enzyme-linked gel electrophoresis, chemiluminescence, in situ hybridization, fluorescence detection, etc.; RNA extraction reagents; reverse transcription reagents; cDNA amplification reagents; standards for preparing standard curves; positive controls. In some embodiments, the reagents for detecting proteins include but are not limited to: blocking solution, antibody diluent, washing buffer, and color development stop solution.

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

[0043] 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;

[0044] 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;

[0045] The output unit outputs the computer diagnosis result or the computer prognosis risk assessment result of the data to be processed.

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

[0047] 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;

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

[0049] 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.

[0050] Furthermore, 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 enhancement algorithm model.

[0051] Furthermore, the diagnostic model calculates the AUC value of each sample and compares the AUC value with the optimal threshold value obtained by the ROC curve generated by the prediction results of the model established based on the ensemble learning method on the test set to diagnose whether the sample has dermatomyositis / antisynthetase syndrome-related interstitial lung disease or is at risk of dermatomyositis / antisynthetase syndrome-related interstitial lung disease.

[0052] Furthermore, the prognostic risk assessment model determines by comparing the level of TREM-1 in each sample that when the expression level of TREM-1 in the sample is greater than or equal to 373.8 ng / ml, the patient's prognostic risk is high, and when the expression level of TREM-1 in the sample is less than 373.8 ng / ml, the patient's prognostic risk is low.

[0053] In some embodiments, the prognostic risk assessment model is used to assess whether dermatomyositis / antisynthetase syndrome-associated interstitial lung disease is a rapidly progressive interstitial lung disease.

[0054] The above is a description of a specific embodiment of the present specification. Other embodiments are within the scope of the appended claims. In some cases, the actions or steps recorded in the claims can be performed in a different order than in the embodiments and still achieve the desired results. In addition, the processes depicted in the accompanying drawings do not necessarily require the specific order or continuous order shown to achieve the desired results. In some embodiments, multitasking and parallel processing are also possible or may be advantageous.

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

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

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

[0058] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0059] Furthermore, the auxiliary materials 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 specifically formulated for administration in fixed or liquid form, including those suitable for administration in the following ways: oral administration, such as medicaments (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those intended for buccal, sublingual and systemic absorption), boluses, powders, granules, pastes for application 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 applied to the skin, lungs or mouth; intravaginal or intrarectal administration, such as pessaries, creams or foams; sublingual administration; ocular administration; transdermal administration; or nasal, pulmonary administration and administration to other mucosal surfaces.

[0061] In the context of the present invention, the term "sample" as used herein refers to a composition obtained or derived from a subject (e.g., an individual of interest) comprising cells and / or other molecular entities to be characterized and / or identified based on, 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 refers to a composition or method of one or more named elements or steps that is open-ended, meaning that the named elements or steps are required, but other elements or steps may be added within the scope of the composition and method. To avoid redundancy, it should also be understood that any composition or method described as "comprising / including" one or more named elements or steps also describes a corresponding more limited composition or method that "consists 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 materially affect the basic and novel characteristics of the composition or method.

[0063] The term "biomarker" refers to a gene or gene product that is a target for regulating one or more target phenotypes (e.g., a target phenotype in a myeloid cell). In this case, the term "biomarker" is synonymous with "target". However, in some embodiments, the term also encompasses measurable entities that have been determined to indicate a target output such as one or more diagnostic, prognostic, and / or therapeutic outputs (e.g., for regulating an inflammatory phenotype, a cancer state, etc.). In other embodiments, the term also encompasses a composition that regulates a gene or gene product, including an anti-gene product antibody and an antigen-binding fragment thereof. Therefore, biomarkers may 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 proposes a novel molecular marker that can be used to predict dermatomyositis / anti-synthetase syndrome combined with interstitial lung disease, especially rapidly progressive interstitial lung disease. The present invention has found through research that the expression of sTREM-1 in the serum of all patients with dermatomyositis / anti-synthetase-related interstitial lung disease, especially patients with rapidly progressive interstitial lung disease, is significantly increased. The serum sTREM-1 content in patients with dermatomyositis / anti-synthetase-related interstitial lung disease reflects to a certain extent 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 combined with interstitial lung disease at the early stage of the disease, and provides an effective technical means for improving the prognosis of patients with dermatomyositis / anti-synthetase syndrome combined with ILD, especially RPILD. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 The figure is the expression result of serum sTREM-1 detected by ELISA in DM / ASS ILD patients; A is the comparison of serum sTREM-1 levels between normal control (HC) population (n=36) and DM / ASS ILD patients (n=67); B is the comparison of serum sTREM-1 levels between normal control (HC) population (n=36), anti-MDA5 antibody positive DM ILD patients (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 2Figure 1 is a result of detecting sTREM-1 levels in the serum of patients with DM / ASS RP ILD; A is a comparison of serum sTREM-1 levels in patients with DM / ASS combined with RP-ILD (n=11), patients without RP-ILD (non-RPILD, n=21) and healthy controls (n=18) in the training set; B is a comparison of serum sTREM-1 levels in patients with DM / ASS combined with RP-ILD (n=13), patients without RP-ILD (non-RPILD, n=22) and healthy controls (n=18) in the validation set; C is an analysis of the expression of TREM-1 in lung tissues of patients with DM / ASS combined with RPILD, without RPILD and normal controls (HC) by immunohistochemical staining; D is a comparison of the expression levels of TREM-1 in lung tissues of patients with DM / ASS combined with RPILD, without RPILD and normal controls (HC); E is a comparison of serum sTREM1 as a marker for distinguishing DM / ASS from DM / ASS. ROC curve analysis of RPILD serum markers between training and test sets; * represents p < 0.05, ** represents p < 0.01; *** represents p < 0.001; scale bar, 50 μm. Arrows, TREM-1 positive cells. AUC represents the area under the ROC curve.

[0068] Figure 3 The following are the results of evaluating the prognosis of DM / ASS ILD patients by serum sTREM-1 levels; A is the comparison of serum sTREM-1 levels between DM / ASS ILD survivors (survivors, n=58) and deceased patients (decedents, n=9); B is the Kaplan-Meier survival analysis of DM / ASS ILD patients grouped according to serum sTREM-1 levels; log rank test p=0.002. DETAILED DESCRIPTION

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

[0070] In practice, receiver operating characteristic curves (ROC curves) are typically calculated by plotting the values ​​of a variable against their relative frequency in a "normal" (e.g., apparently healthy individuals with no prenatal conditions or pathologies) and a "disease" population (similarly, for example, two different conditions such as viral and bacterial RTI or typical and atypical pneumonia). For any particular marker, the distribution of marker levels for subjects with and without the disease / pathology may overlap. Under such conditions, the test cannot absolutely distinguish between normal and disease with 100% accuracy, and the overlapping area may indicate where the test cannot distinguish between normal and disease. A threshold is selected, below which tests are considered "abnormal", above which tests are considered "normal", or below or above which tests indicate a particular pathology. The area under the ROC curve (AUC) is a measure of the probability that a perceptual measurement will allow the correct identification of a pathology. ROC curves can be used even if the test results do not necessarily provide exact numbers. ROC curves can be created as long as the results can be ranked.

[0071] Those skilled in the art will appreciate that any range provided herein also includes all possible sub-ranges and combinations of these sub-ranges, as well as individual values, especially integer values, constituting the range, for any of a variety of purposes, especially for providing textual descriptions. Therefore, it should be understood that each unit between two specific units is also disclosed. For example, if 10 to 15 are disclosed, 11, 12, 13 and 14 are disclosed separately, and as part of the range. The quoted range (such as weight percentage or carbon group) includes each specific numerical value, integer, decimal or characteristic within the range. It should be easily understood that any range listed can fully describe and enable the same range to be decomposed into equal halves, thirds, fourths, fifths or tenths. As a non-limiting example, each range described herein can be easily decomposed into the lower third, the middle third and the upper third, etc. Those skilled in the art will also appreciate that "up to", "at least", "higher than", "lower than", "more than", "greater than", "less than", "or above" or similar terms include the listed numbers, and such terms also refer to the ranges that can be subsequently divided into the sub-ranges mentioned above. Likewise, any ratio listed herein also includes sub-ratios that fall within the broader ratio.

[0072] 1. Research subjects

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

[0074] 2. Clinical data collection

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

[0076] 3. Experimental methods

[0077] 3.1 All serum samples were routinely collected before the patients were treated during hospitalization or outpatient visits and stored at -80°C. The concentration of sTREM-1 was determined using a commercial enzyme-linked immunosorbent assay kit 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. Lung tissue of the control group was obtained from normal tissue during surgical resection of lung adenoma or pulmonary hamartoma. 10% formalin was used to fix the tissue. After dehydration, the fixed tissue was embedded in paraffin (6 μm thickness) and sliced ​​and fully dried. After dewaxing, hydration and antigen retrieval. Endogenous peroxidase was removed with 3% hydrogen peroxide, and after blocking with goat serum, the tissue sections were incubated with anti-TREM-1 polyclonal antibody (1:200 dilution) at 4°C overnight. On the second day, one drop of unlabeled goat anti-mouse / rabbit IgG polymer was added and incubated at room temperature for 30 minutes. After washing, DAB was added for color development and counterstained with hematoxylin. Finally, the tissue was dehydrated and sealed.

[0079] 4. Statistical analysis

[0080] Categorical variables were expressed as counts and percentages, and normally distributed continuous variables were expressed as mean ± standard deviation (SD). The t-test was used for comparison. Non-normally distributed data were expressed as median [interquartile range (IQR)] and compared using the Mann-Whitney U test. The Kruskal-Waills H test was used for comparisons among multiple groups. P < 0.05 was considered statistically significant. SPSS26.0 and GraphPad Prism 8.0 were used for statistical analysis.

[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 only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0082] Example 1

[0083] The expression of sTREM-1 in the serum of 67 DM / ASS patients and 36 age- and sex-matched healthy controls (HC) was preliminarily detected using a commercially available TREM-1 ELISA kit (the kit contains a microplate pre-coated with human TREM-1 specific monoclonal antibody and a human TREM-1 antibody conjugated with horseradish peroxidase) according to the instructions. The results showed that the sTREM-1 level in the serum of DM / ASS ILD patients was significantly higher than that in HC patients ( Figure 1 A). The 67 DM / ASS ILD patients were further divided into anti-melanoma differentiation associated gene 5 (mda5) antibody-positive dermatomyositis-related ILD, ASS-related ILD, and other types of dermatomyositis-related ILD subgroups. Comparison with hc revealed that the serum sTREM1 levels of the three groups of DM / ASS-related ILD patients were not different, but were all higher than those of the hc population ( Figure 1 B). The preliminary results showed that the sTREM-1 level was increased in patients with DM / ASS ILD, and there was no significant difference in different types of dermatomyositis combined with ILD.

[0084] The DM / ASS ILD patients were further divided into rapidly progressive interstitial lung disease (RP ILD) group and 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 AB). Through lung tissue immunohistochemical staining, it was found that the expression of membrane receptor type TREM-1 in lung tissue of RP-ILD group was significantly increased ( Figure 2 C, 2D). This shows that the expression levels of sTREM-1 and membrane receptor form TREM-1 are associated with the severity of DM / ASS ILD. To further clarify this association, we used binary logistic regression analysis to find that increased serum sTREM-1 levels (OR: 1.049; p = 0.020) and 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). Then, the ROC curve was drawn to analyze the predictive value of serum sTREM-1 levels for the diagnosis of RPILD in patients with DM / ASS. The results are shown in Figure 2 As shown in E, the diagnostic threshold of serum sTREM-1 for diagnosing anti-DM / ASS-rpild in the test set was 373.8 ng / ml, with a diagnostic sensitivity of 72.7%, a specificity of 76.2%, an area under the curve of 0.75, and p = 0.004. 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, a long-term follow-up of 67 DM / ASS ILD patients was performed to further clarify the clinical value of serum sTREM-1 in predicting death. As of December 31, 2023, 9 patients died of respiratory failure. Compared with the survivors, the initial serum sTREM-1 levels of the deceased patients were significantly higher ( Figure 3A). The DM / ASS patients were divided into two groups according to whether the initial serum sTREM-1 level was less than 373.8 ng / ml. The results showed that the cumulative survival rate of patients with 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 the 2.5-year cumulative survival rate of patients with sTREM-1 < 373.8 ng / ml was 97.5% ( Figure 3 B).

[0088] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection 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 interstitial lung disease associated with dermatomyositis / antisynthetase syndrome; Preferably, the interstitial lung disease comprises rapidly progressive interstitial lung disease or non-rapidly progressive interstitial lung disease; Preferably, said TREM-1 is soluble TREM-1.

2. The use according to claim 1, wherein the sample comprises bone marrow, blood, blood cells, serum, plasma, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, 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, lavage fluid, aspirate, scraping, bone marrow sample, tissue biopsy sample, surgical sample, secretion and / or excrement; Preferably, the sample includes blood, blood cells, serum, plasma, or bronchoalveolar lavage fluid; Preferably, the subject comprises a human or non-human mammal; Preferably, the subject is a human.

3. The use according to claim 1, wherein the product comprises a reagent, a test kit, a chip or a test paper; Preferably, the product determines whether a subject has dermatomyositis / antisynthetase syndrome-associated interstitial lung disease or is at risk of developing dermatomyositis / antisynthetase syndrome-associated interstitial lung disease by measuring the level of TREM-1 in a sample; Preferably, the reagents include specific primers, probes, antisense oligonucleotides, aptamers or antibodies for detecting biomarkers; Preferably, 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-stranded conformation polymorphism method, allele-specific PCR, SNaPshot method, SNPlex method, denaturing high performance liquid chromatography, and denaturing gradient gel electrophoresis.

4. The use of reagents for detecting the TREM-1 biomarker in samples for the preparation of products for predicting the prognosis of patients with interstitial lung disease associated with dermatomyositis / antisynthetase syndrome; Preferably, the interstitial lung disease comprises rapidly progressive interstitial lung disease or non-rapidly progressive interstitial lung disease; Preferably, said TREM-1 is soluble TREM-1.

5. The use according to claim 4, wherein the sample comprises bone marrow, blood, blood cells, serum, plasma, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, 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, lavage fluid, aspirate, scraping, bone marrow sample, tissue biopsy sample, surgical sample, secretion and / or excrement; Preferably, the sample includes blood, blood cells, serum, plasma, or bronchoalveolar lavage fluid; Preferably, the subject comprises a human or non-human mammal; Preferably, the subject is a human.

6. The use according to claim 4, 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; Preferably, the prognostic risk of the patient with dermatomyositis / antisynthetase 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 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. Preferably, the reagents include specific primers, probes, antisense oligonucleotides, aptamers or antibodies for detecting biomarkers; Preferably, 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-stranded conformation polymorphism method, allele-specific PCR, SNaPshot method, SNPlex method, denaturing high performance liquid chromatography, and denaturing gradient gel electrophoresis.

7. A product for diagnosing dermatomyositis / antisynthetase syndrome-associated interstitial lung disease or predicting the prognosis of a patient with dermatomyositis / antisynthetase syndrome-associated interstitial lung disease, the product comprising a reagent for detecting the gene or protein expression level of the TREM-1 biomarker in a sample; Preferably, the product comprises a reagent, a test kit, a chip or a test paper.

8. 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.

9. The computer system of claim 8, 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.

10. Application of TREM-1 biomarker in screening drugs for the treatment of interstitial lung disease associated with dermatomyositis / antisynthetase syndrome; Preferably, the interstitial lung disease comprises rapidly progressive interstitial lung disease or non-rapidly progressive interstitial lung disease; Preferably, said TREM-1 is soluble TREM-1.

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