Method for predicting clinical outcome of olmaszumab for treating chronic sinusitis with nasal polyp patient according to cytokine baseline expression level in serum and application of method for predicting clinical outcome of olmaszumab for treating chronic sinusitis with nasal polyp patient

Abstract

The invention discloses a method for predicting the clinical outcome of treatment of patients with chronic sinusitis with nasal polyp (CRSwNP) by omphizumab according to the baseline expression level of cytokines in serum and application of the omphizumab. The research finds that the baseline expression levels of CCL-3 and CCL-4 in the serum of a patient can be used for predicting the clinical outcome of SNOT-22 symptom score improvement of a CRSwNP patient treated by aumaszumab for the first time; the baseline expression level of IL-8 in serum can be used for predicting the clinical outcome of nasal obstruction symptom improvement in treatment of a CRSwNP patient by the omaszumab; the baseline expression level of EGF in serum can be used for predicting the clinical outcome of improvement of the facial pain symptom of a CRSwNP patient treated by the omaszumab.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for predicting the clinical outcome of patients with chronic sinusitis and nasal polyps treated with omalizumab according to the baseline expression level of cytokines in serum and its application. Background Art

[0002] Chronic rhinosinusitis (CRS) is a common chronic inflammatory disease involving the sinus mucosa. The course of the disease lasts for more than 12 weeks. The clinical symptoms are mostly nasal congestion, runny nose, facial pain, and hyposmia or anosmia. An epidemiological survey from seven cities in China showed that the prevalence of CRS in China is about 4.8% to 9.7%, with an average prevalence of 8.0%, affecting about 100 million people. [1] .

[0003] CRS can be divided into two types based on clinical phenotype: chronic rhinosinusitis with nasal polyps (CRSwNP) and chronic rhinosinusitis without nasal polyps (CRSsNP). [2] Although the phenotypic classification of CRS has been established, both CRS can be further divided into different endotypes based on the underlying immune type and cell differentiation pattern. [3] .

[0004] In general, CRSwNP patients have more severe clinical symptoms, a relatively high recurrence rate, and a high comorbidity rate of diseases such as bronchial asthma, aspirin-exacerbated respiratory diseases, and allergic rhinitis. From the perspective of immune endotype, CRS can be divided into type 1 (type 1) represented by interferon-(IFN-), type 2 (type 2) represented by IL-4, IL-5, and IL-13, and type 3 (type 3) CRS represented by the IL-17 family, according to different inflammatory factor driving modes. CRSsNP is usually dominated by type 1 and type 3 inflammation, and CRSwNP is dominated by eosinophil infiltration and type 2 inflammation. [4] Although the “one-to-two” phenotypic classification method is still generally applicable to clinical treatment, the endotype study of CRS reflects the complexity of the pathogenesis of CRS, especially the mixed inflammatory pattern that often occurs in refractory CRS. [5-7] Type 2 CRS plays a decisive role in the patient's quality of life, disease severity, and disease prognosis [8] .

[0005] Omalizumab is a biologic agent for type 2 CRS, but its clinical outcomes in patients with CRSwNP vary [9] .

[0006] The Chinese patent application number 202311274974.4, "A method and application for predicting or evaluating the efficacy of omalizumab in the treatment of recurrent nasal polyps using local IgE", discloses that: the high expression level of total IgE in nasal secretions may predict poor clinical outcomes in patients with CRSwNP treated with omalizumab. ① According to the EPOS2020 guidelines and EUFOREA's recommendations, the first evaluation time for biological agents is 6 months after treatment. The evaluation time after omalizumab treatment in this patent is 16 weeks. The short evaluation time may lead to inaccurate evaluation results; ② The effective evaluation indicator confirmed by this patent is local IgE, and the sampling method is to clamp nasal polyp tissue, which is an invasive operation; ③ The processing of nasal secretions also requires centrifugation and standardized quantitative processing of BCA, and judgment after immunostaining, which is more complicated; ④ In addition, the expression of different inflammatory factors in patients varies greatly, and the expression level of only one cytokine may not be enough to fully explain the patient's disease level. ⑤In addition, this patent only uses NPS improvement of more than 1 point as the evaluation criterion, while the SNOT-22 score judges the patient's response effect from multiple dimensions, and the evaluation angle is more comprehensive.

[0007] Research progress on omalizumab in the treatment of chronic rhinosinusitis with nasal polyps

[10] Reported: Mortuaire et al. studied and analyzed the eosinophil counts, IgE, IL-5, IgA, ECP and other factors in the blood, nasal secretions and nasal polyps of 57 patients with CRSwNP. Except for the serum total IgE level in the allergic group, which was higher than that in the control group, there were no significant differences in other levels. This article is a general review of the effectiveness of omalizumab in the treatment of chronic sinusitis with nasal polyps, and did not propose its potential effective indicators for predicting clinical prognosis.

[0008] Research progress on omalizumab targeted therapy for chronic sinusitis with nasal polyps

[11] It was reported that in CRSwNP, the epithelial cells (ECs) of the sinuses are the first line of defense to protect the nasal cavity. In nasal polyps, ECs can trigger a type 2 inflammatory response, and the epithelial barrier of the nasal and sinonasal mucosa is destroyed and lacks structural integrity, resulting in an active epithelial cell inflammatory state. ECs can produce IL-33, IL-1, thymic stromal lymphopoietin (TSLP), and IL-25 to respond to various damage-related substances, such as pathogenic microorganisms and their related products or allergens. Superantigens from the nasal cavity, Staphylococcus aureus colonies, can lead to the activation of polyclonal T cells and B cells, thereby amplifying the inflammatory response. Epithelial cytokines such as TSLP, IL-25, and IL-33 induce the activation and accumulation of type 2 innate lymphocytes (ILC2), leading to the production of IL-5, IL-13, and IL-4. At the same time, helper T cell 2 (Th2) cells can directly respond to IL-25 and IL-33, further amplifying the inflammatory response. This article is a general review of the effectiveness of omalizumab in the treatment of chronic sinusitis with nasal polyps, and does not propose its potential effective indicators for predicting clinical prognosis.

[0009] Currently, there is no method with high accuracy to predict the clinical outcomes of patients with chronic sinusitis and nasal polyps treated with omalizumab. Establishing a method to predict the clinical outcomes of patients with chronic sinusitis and nasal polyps treated with omalizumab is of great significance in guiding the clinical use of omalizumab.

[0010] References:

[0011] [1] Bachert C, Marple B, Schlosser RJ, et al. Adult chronicrhinosinusitis. Nat Rev Dis Primers. 2020; 6(1):86.

[0012] [2]Shi JB, Fu QL, Zhang H, et al. Epidemiology of chronic rhinosinusitis: results from a cross-sectional survey in seven Chinese cities. Allergy.

[0013] 2015;70(5):533-539.

[0014] [3]Klingler AI,Stevens WW,Tan BK,et al.Mechanisms and biomarkers ofinflammatory endotypes in chronic rhinosinusitis without nasal polyps.JAllergy Clin Immunol.2021;147(4):1306-1317.

[0015] [4]Bachert C,Zhang N,Hellings PW,Bousquet J.Endotype-driven carepathways in patients with chronic rhinosinusitis.J Allergy Clin Immunol.2018;141(5):1543-1551.

[0016] [5]Cho SH,Kim DW,Gevaert P.Chronic Rhinosinusitis without NasalPolyps.J Allergy Clin Immunol Pract.2016;4(4):575-582.

[0017] [6]Tomassen P,Vandeplas G,Van Zele T,et al.Inflammatory endotypes ofchronic rhinosinusitis based on cluster analysis of biomarkers.J Allergy ClinImmunol.2016;137(5):1449-1456.e1444.

[0018] [7]Kato A,Schleimer RP,Bleier BS.Mechanisms and pathogenesis ofchronic rhinosinusitis.J Allergy Clin Immunol.2022;149(5):1491-1503.

[0019] [8]Wang

[0020] [9] Fokkens WJ, Lund VJ, Hopkins C, et al. European Position Paper on Rhinosinusitis and Nasal Polyps 2020. Rhinology. 2020; 58 (Suppl S29): 1-464.

[0021]

[10] Wang Aowei, Shi Wenjie. Research progress of omalizumab in the treatment of chronic rhinosinusitis with nasal polyps[J]. International Journal of Otorhinolaryngology Head and Neck Surgery, 2021, (Issue 4).

[0022]

[11] Zhang Yunqiu, Ren Xiumin, Xu Ou, Dong Jinhui, Wang Jianxing. Research progress of omalizumab targeted therapy for chronic sinusitis with nasal polyps [J / OL]. Journal of Otolaryngology and Ophthalmology of Shandong University. https: / link.cnki.net / urlid / 37.1437.R.20240408.1519.006. Summary of the invention

[0023] In view of the problems existing in predicting the clinical outcomes of patients with chronic rhinosinusitis and nasal polyps (CRSwNP) treated with omalizumab at this stage, the present application detects the baseline expression levels of 48 cytokines in the serum of patients before omalizumab treatment (only 36 cytokines can be detected). 24 weeks after omalizumab treatment, the clinical outcomes of patients are evaluated by three methods: SNOT-22, NCS and FPS scores. It is found that the expression levels of CCL-3 and CCL-4 in serum can predict the clinical outcomes of omalizumab treatment of CRSwNP patients with improved SNOT-22 symptom scores; the expression level of IL-8 in serum can predict the clinical outcomes of omalizumab treatment of CRSwNP patients with improved nasal congestion symptoms; the expression level of EGF in serum can predict the clinical outcomes of omalizumab treatment of CRSwNP patients with improved facial pain symptoms. To achieve the above objectives, the specific technical scheme of the present invention is as follows:

[0024] The first aspect: basic data and research design

[0025] A total of 16 patients with chronic sinusitis and nasal polyps aged 18 years and above and 16 patients aged 18 years and above who required nasal expansion surgery due to nasal septum deviation participated in this study. The basic characteristics of the patients were collected, including gender, age, BMI, asthma, allergic rhinitis, VAS score of nasal polyps, VAS score of nasal congestion, VAS score of runny nose, VAS score of olfaction, VAS score of facial tenderness, SNOT-22 score, and peripheral blood eosinophil count.

[0026] Patients receiving omalizumab treatment will receive the required subcutaneous injection dose according to the patient's baseline body weight (kg) and serum total IgE (kU / L) in accordance with the instructions.

[0027] Second, detect the expression levels of 48 cytokines in patient serum

[0028] Before omalizumab treatment, samples were taken to detect the baseline expression levels of 48 cytokines in the patients' serum; samples were taken to detect the expression levels of 48 cytokines in the patients' serum at 24 weeks of treatment.

[0029] 44 cytokines including IL-1α, IL-1β, IL-1Rα, and IL-2 were detected by Luminex;

[0030] MPO and periostin in serum were detected by enzyme-linked immunosorbent assay (Elisa);

[0031] The expression levels of eosinophil cationic protein (ECP) and total IgE were automatically detected by the ImmunoCAP system.

[0032] The third aspect is the evaluation index of clinical symptom improvement

[0033] The nasosinusitis outcome test 22 (SNOT-22), nasal congestion score (NCS) and facial pain score (FPS) of the enrolled patients were collected at baseline, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks and 24 weeks after omalizumab administration. The improvement of SNOT-22 score by more than 8.9 points, NCS score by more than 2 points or FPS score by more than 2 points was considered as treatment improvement, and the patients after treatment were divided into treatment improvement group and treatment suboptimal group.

[0034] Fourthly, differentially expressed cytokines and predictive efficacy in the serum of patients in the treatment improvement group and the poor treatment group

[0035] result:

[0036] In the treatment improvement group and the poor treatment group divided by SNOT-22 score improvement >8.9 points, the ROC-AUC of serum CCL-3 and CCL-4 expression levels in predicting the clinical outcome of SNOT-22 symptom score improvement in CRSwNP patients treated with omalizumab were 0.820 and 0.900, respectively;

[0037] The treatment improvement group and the poor treatment group were divided by NCS score improvement of >2 points. The ROC-AUC of serum IL-8 expression level in predicting the clinical outcome of nasal congestion symptom improvement of CRSwNP patients treated with omalizumab was 0.883.

[0038] The treatment improvement group and the poor treatment group were divided by FPS score improvement of >2 points. The ROC-AUC of EGF expression level in serum for predicting the clinical outcome of improvement of facial pain symptoms in patients with CRSwNP treated with omalizumab was 0.810.

[0039] Fifth aspect: Verify the predictive efficacy of the four cytokines in the fourth aspect

[0040] result:

[0041] The treatment improvement group and the poor treatment group were divided into two groups with SNOT-22 score improvement of >8.9 points. The expression levels of CCL-3 and CCL-4 in serum had good accuracy, sensitivity and specificity in predicting the clinical outcome of SNOT-22 symptom score improvement in patients with CRSwNP treated with omalizumab, and could be used to predict clinical outcomes.

[0042] The treatment improvement group and the poor treatment group were divided by NCS score improvement of >2 points. The expression level of serum IL-8 had good accuracy, sensitivity and specificity in predicting the clinical outcome of improvement of nasal congestion symptoms in CRSwNP patients treated with omalizumab, and could be used to predict clinical outcomes.

[0043] The treatment improvement group and the poor treatment group were divided by an FPS score improvement of >2 points. The expression level of EGF in serum had good accuracy, sensitivity and specificity in predicting the clinical outcome of improvement of facial pain symptoms in patients with CRSwNP treated with omalizumab, and could be used to predict clinical outcomes.

[0044] Compared with the prior art, the beneficial effects of this application are:

[0045] This application first discovered:

[0046] 1. The baseline expression levels of CCL-3 and CCL-4 in serum have good accuracy, sensitivity and specificity in predicting the clinical outcome of improvement in SNOT-22 symptom scores in patients with CRSwNP treated with omalizumab, and can be used to predict the clinical outcome after omalizumab treatment;

[0047] 2. The baseline expression level of IL-8 in serum has good accuracy, sensitivity and specificity in predicting the clinical outcome of improvement of nasal congestion symptoms in patients with CRSwNP treated with omalizumab, and can be used to predict the clinical outcome after omalizumab treatment;

[0048] 3. The baseline expression level of EGF in serum has good accuracy, sensitivity and specificity in predicting the clinical outcomes of CRSwNP patients treated with omalizumab, and can be used to predict the clinical outcomes of improvement in facial pain symptoms after omalizumab treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 , Cytokines differentially expressed in serum between the treatment improvement group and the suboptimal treatment group of CRSwNP patients treated with omalizumab,

[0050] A is the statistical diagram of the difference in serum CCL-3 expression between the treatment improvement group and the poor treatment group, divided by SNOT-22 score improvement>8.9 points.

[0051] B is the statistical diagram of the difference in serum CCL-4 expression between the treatment improvement group and the poor treatment group, divided by SNOT-22 score improvement>8.9 points.

[0052] C is the statistical diagram of the difference in serum IL-8 expression between the treatment improvement group and the poor treatment group divided by NCS score improvement > 2 points,

[0053] D is the statistical diagram of the difference in serum EGF expression between the improved treatment group and the poorly treated group, divided by FPS score improvement of >2 points;

[0054] Figure 2 , ROC curve of four cytokines in serum predicting the clinical outcomes of omalizumab in the treatment of chronic sinusitis with nasal polyps, among which,

[0055] A is the treatment improvement group and the poor treatment group divided by SNOT-22 score improvement>8.9 points. The ROC curve of serum CCL-3 and CCL-4 predicting the clinical outcome of omalizumab treatment of chronic sinusitis with nasal polyps.

[0056] B is the ROC curve of the treatment improvement group and the poor treatment group divided by NCS score improvement of >2 points, and the serum IL-8 predicts the clinical outcome of omalizumab in the treatment of chronic sinusitis with nasal polyps.

[0057] C is the ROC curve of the treatment improvement group and the poor treatment group divided by FPS score improvement of >2 points, and the serum EGF predicts the clinical outcome of omalizumab in the treatment of chronic sinusitis with nasal polyps.

[0058] Figure 1 middle,

[0059] *, **, *** and **** represent P<0.05, P<0.01, P<0.001 and P<0.0001, respectively, indicating that the difference between the two groups was statistically significant. DETAILED DESCRIPTION

[0060] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0062] All materials, reagents, etc. in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0063] Reagents and Materials

[0064]

[0065]

[0066]

[0067] instrument

[0068]

[0069] Example 1. Patient basic information and study design

[0070] This study was a single-center trial conducted at Beijing Tongren Hospital from January 2019 to January 2023.

[0071] A total of 16 patients aged 18 years and above with chronic sinusitis and nasal polyps and 16 patients aged 18 years and above who required nasal expansion surgery due to nasal septum deviation participated in this study.

[0072] This study was approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University (TRECKY2019-070). Written informed consent was obtained before patient participation.

[0073] Thirty-two patients were divided into an omalizumab treatment group (n=16) and a control group (n=16). Patients receiving omalizumab treatment will receive the required subcutaneous injection dose according to the patient's body weight (kg) and serum total IgE (kU / L) at baseline according to the standards of the instructions. During the observation period, patients were allowed to use nasal spray glucocorticoids, antihistamines and antihistamines to treat nasal diseases, and were advised to use bronchial asthma treatment drugs regularly. The basic characteristics of the patients are shown in Table 1.

[0074]

[0075]

[0076] Note: *: P<0.05

[0077] Example 2. Evaluation indicators for improvement of clinical symptoms

[0078] Demographic data were collected from all patients. 22-item Sino-Nasal Outcome Test (SNOT-22) was collected from the enrolled patients at baseline, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, and 24 weeks; nasal congestion score (NCS) and facial pain score (FPS) were used for further analysis.

[0079] A SNOT-22 score of >8.9 before and after treatment was considered improvement.

[0080] NCS and FPS scores before and after treatment>2 points were considered to be improved.

[0081] The total SNOT-22 score is 110 points, with 0 indicating no symptoms and 110 indicating very severe symptoms.

[0082] The NCS and FPS scores are based on a 10-point system, with 0 indicating no symptoms and 10 indicating very severe symptoms.

[0083] An improvement of more than 8.9 points in SNOT-22 score, more than 2 points in NCS score, or more than 2 points in FPS score was considered as treatment improvement. After treatment, the patients were divided into a treatment improvement group and a poor treatment group. The number of patients in the treatment improvement group and the poor treatment group divided by the improvement of SNOT-22, NCS, and FPS scores at 24 weeks are shown in Table 1.

[0084] Table 1. Number of cases in the treatment improvement group and the treatment suboptimal group according to SNOT-22, NCS and FPS scores at week 24

[0085] Treatment improvement group (cases) Poorly treated group (cases) SNOT-22 score improvement greater than 8.9 points 11 5 NCS score improved by more than 2 points 10 6 FPS score improved by more than 2 points 7 9

[0086] Example 3: Detection of baseline expression levels of 48 cytokines in patient serum

[0087] 1. Sampling

[0088] Before and 24 weeks of omalizumab treatment, 4 mL of blood was collected from the patient's vein in a separation gel vacuum tube. After collection, the tube was placed upright on a test tube rack, left to stand at room temperature for 30 minutes, and then centrifuged at 4°C and 1800g for 15 minutes within 90 minutes. The upper layer of serum was stored in a 1.5 mL transparent sterile centrifuge tube and stored in a -80°C refrigerator.

[0089] 2. Detection

[0090] 2.1 Luminex detection

[0091] The expression levels of 44 cytokines in the serum of patients at baseline and after 24 weeks of omalizumab treatment were detected by Luminex, including interleukin (IL)-1α, IL-1β, IL-1Rα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-25, IL-33, CD40L, epidermal growth factor (EGF), eotaxin, basic fibroblast growth factor (FGF-basic), FMS-like tyrosine kinase receptor 3 ligand, Flt-3 Ligand, granulocyte colony-stimulating factor, leukocyte antigen 2 (LEC), pro-inflammatory cytokine, and leukocyte antigen 2 (P2P). factor, G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), granzyme B, chemokine ligand-1 (CXC motif, CXCL-1), CXCL-2, interferon (interferon, IFN)-α, IFN-β, IFN-γ, IP-10, chemokine (C-C motif) ligand (CCL)-2, CCL-3, CCL-4, CCL-5, CCL-19, CCL-20, programmed death receptor-1 (cell death ligand 1, PD-L1), platelet-derived growth factor (PDGF)-AA, PDGF-AB, transforming growth factor (TGF)-α, tumor necrosis factor (TNF)-α, tumor necrosis factor-related apoptosis-inducing ligand (TNF-related The main inhibitors of TNF-α and TNF-α are TRAIL, apoptosis-inducing ligand and vascular endothelial growth factor (VEGF).

[0092] (1) Before the experiment, restore the Human XL Cytokine Fixed Panel (RD#LKTM04 and RD#LKTM014B) kit to room temperature; prepare the standard, washing solution, quality control, mixed microsphere solution, Biotin-antibody solution and PE-streptavidin solution according to the instructions;

[0093] (2) Prepare the plate layout and add 50 μL of standard or sample to be tested to each well;

[0094] (3) Add 50 μL of resuspended magnetic beads to each well, seal the plate with aluminum foil, incubate at room temperature for 2 h, and adjust the shaking speed to 800 rpm;

[0095] (4) Add the magnetic plate to the bottom of the microplate, let it stand for 1 min, discard the liquid, take the magnetic plate, add 100 μL of washing solution to each well, let it stand for 1 min, add the magnetic plate to the bottom of the microplate again, let it stand for 1 min, and discard the liquid. Repeat the above washing steps 3 times;

[0096] (5) Add 50 μL of Biotin-antibody to each well, seal the plate with aluminum foil, incubate at room temperature for 1 h, and adjust the shaking speed to 800 rpm;

[0097] (6) Add the magnetic plate to the bottom of the microplate, let it stand for 1 min, discard the liquid, take the magnetic plate, add 100 μL of washing solution to each well, let it stand for 1 min, add the magnetic plate to the bottom of the microplate again, let it stand for 1 minute, and discard the liquid. Repeat the above washing steps 3 times;

[0098] (7) Add 50 μL of PE-streptavidin to each well, seal the plate with aluminum foil, incubate at room temperature for 30 min, and adjust the shaking speed to 800 rpm;

[0099] (8) Add the magnetic plate to the bottom of the microplate, let it stand for 1 min, discard the liquid, take the magnetic plate, add 100 μL of washing solution to each well, let it stand for 1 min, add the magnetic plate to the bottom of the microplate again, let it stand for 1 min, and discard the liquid. Repeat the above washing steps 3 times;

[0100] (9) Add 100 μL of washing solution to each well and shake at 800 rpm for 2 min at room temperature to suspend the magnetic beads;

[0101] (10) Read the results on a Luminex 200 instrument within 90 min.

[0102] 2.2 Enzyme-linked immunosorbent assay (ELISA) detection

[0103] Serum MPO and periostin were detected by enzyme-linked immunosorbent assay (ELISA).

[0104] (1) Before the experiment, equilibrate the Elisa kit to room temperature according to the instructions, prepare the standard and washing solution according to the instructions, and prepare the nasal secretions and serum samples to be tested;

[0105] (2) Prepare the plate layout and add 100 μL of RD1-27 to each well;

[0106] (3) Within 15 min, add 50 μL of standard or sample to be tested to each well, seal the plate, and incubate at room temperature for 2 h with the shaker set to 500 rpm;

[0107] (4) Remove the liquid in the wells, add 400 μL of washing solution to each well, soak for 1 to 2 min, remove and tap the liquid in the wells, and wash the plate 4 times in total;

[0108] (5) Add 200 μL of MPO conjugate to each well, seal the plate, incubate at room temperature for 2 h, and adjust the shaking speed to 500 rpm;

[0109] (6) Remove the liquid in the wells, add 400 μL of washing solution to each well, soak for 1 to 2 hours, remove and tap the liquid in the wells, and wash the plate 4 times in total;

[0110] (7) Add 200 μL of substrate solution to each well, seal the plate, and incubate at room temperature in the dark for 30 min;

[0111] (8) Add 50 μL of stop solution to each well and gently shake to ensure thorough mixing;

[0112] (9) Immediately measure the OD450-540 values ​​using a microplate reader;

[0113] (10) Calculate the protein concentration in the sample based on the standard curve.

[0114] 2.3 ImmunoCAP Phadiatop detection

[0115] The expression levels of eosinophil cationic protein (ECP) and total IgE were automatically detected by the ImmunoCAP system.

[0116] Turn on the Phadia 1000 instrument system and Prime software, and the instrument automatically enters the ISW software interface. Click "LOAD REAGENTS" in the ISW software, add and replenish relevant reagents and consumables (Wash Buffer, substrate solution, stop solution, enzyme-labeled secondary antibody, curve control or calibration products, etc.) according to the instrument menu, start the instrument for detection, and check the result data after the detection is completed.

[0117] 3. Statistical methods

[0118] Data analysis was performed using IBM SPSS 23.0 software (SPSS, USA) and GraphPad Prism 9 software (GraphPad Software, USA). Baseline characteristics of patients were expressed as mean ± SD or median-interquartile range (IQR) for continuous variables; Kolmogorov-Smirnov test was used to assess the normal distribution of continuous variable data; Fisher exact test or Chi-squared test was used to compare gender, residential area, education level, smoking status, comorbidities, and monthly income between the two groups. Mann-Whitney Utest was used for intergroup comparison of continuous variables, that is, to compare age, BMI, and cytokine expression levels between the two groups. Wilcoxon signed rank test was used for paired sample test, and Bonferroni correction was used for multiple testing. Spearman correlation analysis was used to evaluate the correlation between biomarkers and the degree of improvement of clinical symptoms, and the BH (Benjamini-Hochberg) method was used for multiple testing correction. ROC curve (Receiveroperating characteristic curve) was used to evaluate the ability of different clinical or cytokine factors to predict the response to omalizumab treatment of CRSwNP. Youden index determined the optimal cutoff value and was used to calculate sensitivity and specificity. P < 0.05 was considered statistically significant.

[0119] Example 4: Differentially expressed cytokines in the serum of patients in the treatment improvement group and the poor treatment group

[0120] 1. Expression levels of 48 baseline cytokines in serum of patients in the treatment improvement group and the poor treatment group

[0121] The expression levels of 48 baseline cytokines were detected (only 36 cytokines could be detected). The cytokines that were not detected were: IL-2, IL-3, IL-4, IL-5, IL-12, IL-13, IL-17, IL-25, Granzyme B, IFN-a, IFN-g, and PDGF-AA.

[0122] According to the improvement of clinical symptoms after treatment, the patients were divided into a treatment improvement group and a poor treatment group using SNOT-22 score improvement of more than 8.9 points, NCS score improvement of more than 2 points, and FPS score improvement of more than 2 points, respectively. The differentially expressed cytokines were analyzed. The results are shown in Tables 2-4.

[0123] Table 2. Baseline cytokine expression levels in CRSwNP patients grouped according to improvement in SNOT-22 score of more than 8.9 points

[0124]

[0125]

[0126]

[0127] Table 3. Baseline cytokine expression levels in CRSwNP patients grouped according to NCS score improvement of more than 2 points

[0128]

[0129]

[0130]

[0131] Table 4. Baseline cytokine expression levels in CRSwNP patients grouped according to FPS improvement of more than 2 points

[0132]

[0133]

[0134]

[0135] 2. Differentially expressed cytokines

[0136] As can be seen from Table 2-4, in the serum of patients in the treatment improvement group and the poor treatment group, the SNOT-22 score improved by more than 8.9 points, and the differentially expressed cytokines were CCL-3 and CCL-4; the NCS score improved by more than 2 points, and the differentially expressed cytokines were IL-8; the FPS score improved by more than 2 points, and the differentially expressed cytokine was EGF.

[0137] Example 4: Prediction performance

[0138] The receiver operating characteristic curve (ROC) analysis was performed on the differentially expressed cytokines screened in Example 3, and the area under the curve (AUC) was calculated. The accuracy of differentially expressed cytokines in predicting the clinical outcomes of patients with chronic sinusitis and nasal polyps treated with omalizumab, as well as their sensitivity and specificity, were evaluated by AUC, Cutoff and Youden Index. The results are shown in Tables 5 and Figure 2 .

[0139] Table 5. Accuracy, sensitivity, and specificity of serum biomarkers in predicting clinical outcomes in patients with CRS with nasal polyps treated with omalizumab

[0140]

[0141] in conclusion:

[0142] The expression levels of CCL-3 and CCL-4 in serum can predict the clinical outcome of omalizumab treatment for the improvement of SNOT-22 symptom scores in patients with CRSwNP;

[0143] The expression level of IL-8 in serum can predict the clinical outcome of improvement of nasal congestion symptoms in patients with CRSwNP treated with omalizumab;

[0144] The expression level of EGF in serum can predict the clinical outcome of improvement of facial pain symptoms in patients with CRSwNP treated with omalizumab.

[0145] Example 5: Verification of prediction performance

[0146] 1. Sample collection

[0147] A total of 16 patients with CRS aged 18 years and above participated in this study.

[0148] The rest is the same as Example 1.

[0149] 2. Evaluation indicators of clinical symptom improvement

[0150] See Example 2 for the method.

[0151] 3. The method for detecting the expression levels of four cytokines, CCL-3, CCL-4, IL-8, and EGF in the patient's plasma is shown in Implementation 3.

[0152] 4. Verify the prediction performance

[0153] Receiver operating characteristic (ROC) curve analysis was performed, and the area under the curve (AUC) was calculated. The accuracy of differentially expressed cytokines in predicting the clinical outcomes of patients with CRS with nasal polyps treated with omalizumab, as well as their sensitivity and specificity, were evaluated by AUC, Cutoff, and Youden index.

[0154] 5. Results and Conclusions

[0155] The expression levels of CCL-3 and CCL-4 in serum have good accuracy, sensitivity and specificity in predicting the clinical outcome of improvement of SNOT-22 symptom score in patients with CRSwNP treated with omalizumab, and can be used to predict the clinical outcome of patients;

[0156] The expression level of IL-8 in serum has good accuracy, sensitivity and specificity in predicting the clinical outcome of improvement of nasal congestion symptoms in patients with CRSwNP treated with omalizumab, and can be used to predict the clinical outcome of patients;

[0157] The expression level of EGF in serum has good accuracy, sensitivity and specificity in predicting the clinical outcome of improvement of facial pain symptoms in patients with CRSwNP treated with omalizumab, and can be used to predict the clinical outcome of patients.

[0158] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Use of a reagent for detecting the baseline expression level of either CCL-3 or CCL-4 in the serum of patients with chronic sinusitis and nasal polyps in the preparation of a product for predicting the clinical outcome of improvement in SNOT-22 symptom score after omalizumab treatment, characterized in that: The SNOT-22 is the Nasal Sinus Outcome Test 22, and the treatment improvement evaluation indicator of the clinical outcome is an improvement in the SNOT-22 score greater than 8.9 points.

2. The use according to claim 1, characterized in that Patients who improved with omalizumab had significantly higher baseline serum CCL-3 expression levels compared with those who did not respond well to omalizumab.

3. The use according to claim 1, characterized in that Patients who improved with omalizumab had significantly higher baseline serum CCL-4 expression levels compared with those who did not respond well to omalizumab.

4. Use of a reagent for detecting the baseline expression level of IL-8 in the serum of patients with chronic sinusitis and nasal polyps in the preparation of a product for predicting the clinical outcome of improvement of nasal congestion symptoms after omalizumab treatment, characterized in that: The treatment improvement evaluation indicator of the clinical outcome is an improvement in the nasal congestion score (NCS) of more than 2 points.

5. The use according to claim 1, characterized in that Patients who improved with omalizumab had significantly lower baseline serum IL-8 expression levels compared with those who did not respond well to omalizumab.

6. Use of a reagent for detecting the baseline expression level of EGF in the serum of patients with chronic sinusitis and nasal polyps in the preparation of a product for predicting the clinical outcome of improvement of facial pain symptoms after omalizumab treatment, characterized in that: The evaluation indicator of the treatment improvement of the clinical outcome is an improvement of the facial pain score FPS by more than 2 points.

7. The use according to claim 1, characterized in that Patients who improved on treatment had significantly lower baseline serum EGF expression levels compared with patients who did not respond well to omalizumab.

8. The use according to any one of claims 1, 4 or 6, characterized in that: The products include test kits, chips, and test strips.

9. The use according to any one of claims 1, 4 or 6, characterized in that: The CCL-3, CCL-4, IL-8 and EGF were detected by Luminex.

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