Biomarker for predicting children chronic sinusitis with nasal polyp relapse and application thereof

Abstract

The invention belongs to the field of medical diagnosis, and particularly provides a biomarker for predicting children chronic sinusitis with nasal polyp relapse and application of the biomarker. The invention discloses the biological marker ITGAM and / or FCGR3B related to children chronic sinusitis with nasal polyp relapse for the first time, and experiments prove that whether a child patient suffers from nasal polyp relapse or not can be accurately predicted by detecting the expression condition of ITGAM and / or FCGR3B in nasal polyp tissues of the child patient suffering from chronic sinusitis with nasal polyp, so that the risk of relapse of nasal polyp is reduced, and the risk of relapse of nasal polyp is reduced. Therefore, the follow-up visit frequency and medication after the operation can be adjusted according to the recurrence probability of nasal polyp of the sick child, and the method has extremely high guidance value for postoperative prognosis of the sick child.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis, and in particular, to a biomarker for predicting the recurrence of chronic sinusitis with nasal polyps in children and an application thereof. Background Art

[0002] Chronic rhinosinusitis (CRS) is a highly heterogeneous disease associated with chronic inflammation of the nasal mucosa. In adults, the prevalence of CRS based on a combination of symptoms and endoscopy or CT scans is 3-6%. In children, the prevalence is as high as 4%. CRS is often difficult to diagnose in children, and the incidence of complications is high, which has a serious impact on the quality of life of children and their parents and imposes a heavy economic burden on society.

[0003] A major challenge facing pediatric CRSwNP in clinical practice is its high recurrence rate. Although the development of endoscopic sinus surgery has significantly improved the treatment effect, postoperative recurrence remains a thorny problem. Using standard functional endoscopic sinus surgery, the recurrence rate is still as high as 10-35%. The recurrence of CRSwNP involves many factors, including insufficient resection during the initial surgery, maxillary sinus choanal polyps, and cystic fibrosis, and the risk of recurrence is still difficult to accurately quantify. The recurrence of CRSwNP not only affects the physical health of children, but may also have a serious impact on their learning and social abilities. It also means a heavier financial burden on the family and occupies more medical resources in society. Summary of the invention

[0004] In order to fill the gap in the prior art, the present invention provides a biomarker for predicting the recurrence of chronic sinusitis with nasal polyps in children and its application.

[0005] First, in a first aspect, the present invention provides a biomarker for predicting the recurrence of chronic sinusitis with nasal polyps in children, wherein the biomarker is ITGAM and / or FCGR3B.

[0006] The second aspect of the present invention provides a use of a reagent for detecting the expression level of the above-mentioned biomarker in the preparation of a kit for predicting the recurrence of chronic sinusitis with nasal polyps in children.

[0007] In a preferred embodiment, the reagent comprises an immunohistochemical staining reagent.

[0008] In a more preferred embodiment, the method of using the kit is: detecting the expression levels of ITGAM and FCGR3B in polyp tissues of children with chronic sinusitis and nasal polyps; if 1-2 of ITGAM and FCGR3B are positive, the child is judged to have a high risk of nasal polyp recurrence and needs to increase the frequency of postoperative follow-up and medication.

[0009] The third aspect of the present invention provides a kit for predicting the recurrence of chronic sinusitis with nasal polyps in children, wherein the kit comprises a reagent for detecting the expression level of the above-mentioned biomarkers.

[0010] In a preferred embodiment, the reagent comprises an immunohistochemical staining reagent.

[0011] In a more preferred embodiment, the method of using the kit is: detecting the expression levels of ITGAM and FCGR3B in polyp tissues of children with chronic sinusitis and nasal polyps; if 1-2 of ITGAM and FCGR3B are positive, the child is judged to have a high risk of nasal polyp recurrence and needs to increase the frequency of postoperative follow-up and medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 Bubble charts for GO and KEGG enrichment analysis of DEGs, where the left chart shows the TOP20 enriched entries of DEGs in biological processes, and the right chart shows the KEGG pathways where DEGs are significantly enriched; p -values ​​< 0.05 and q -values ​​< 0.2 were considered statistically significant; Figure 2 Screening for PPI networks and hub genes of DEGs; Figure 3 is the RNA expression level graph of hub genes; Figure 4 The expression of FN1, CXCL8, EGF, ITGAX, ITGAM, FCGR3B, and IFNG at the protein level; Figure 5 To compare the sinus CT images of multiple recurrent patients before the first operation and at the time of recurrence, it can be seen whether the first preoperative NP was localized or diffuse, and the recurrence was manifested as NP originating from the maxillary sinus; from top to bottom, the pictures are: patient A 20190618 first operation CT image, patient A 20220517 second operation CT image, patient B 20210825 first operation CT image, patient B 20220712 second operation CT image, patient C 20210824 first operation CT image, patient C 20220719 second operation CT image; Figure 6 There were no significantly different expression genes between localized CRSwNP and recurrent CRSwNP; Figure 7The protein expressions of ITGAM and FCGR3B genes in localized and recurrent CRSwNP; Figure 8 Figure 2 ROC curves of PCR and immunohistochemistry results of ITGAM and FCGR3B in localized and recurrent CRSwNP, including (a) ROC curve of PCR results of ITGAM in localized CRSwNP; (b) ROC curve of PCR results of FCGR3B in localized CRSwNP; (c) ROC curve of PCR results of ITGAM in recurrent CRSwNP; (d) ROC curve of PCR results of FCGR3B in recurrent CRSwNP; (e) ROC curve of immunohistochemistry results of ITGAM in localized CRSwNP; (f) ROC curve of immunohistochemistry results of FCGR3B in localized CRSwNP; (g) ROC curve of immunohistochemistry results of ITGAM in recurrent CRSwNP; (h) ROC curve of immunohistochemistry results of FCGR3B in recurrent CRSwNP. DETAILED DESCRIPTION

[0013] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0014] The English abbreviations and Chinese definitions in the following examples are: Chronic rhinosinusitis: CRS Nasal polyps: NP Chronic Rhinosinusitis with Nasal Polyps: CRSwNP Chronic Rhinosinusitis without Nasal Polyps: CRSsNP Integrin αM: ITGAM IgG-Fc fragment low affinity receptor IIIb: FCGR3B In the following examples, all data were analyzed using Graphad Prism9 (Version 9.3.1) software. Continuous variables were tested for normal distribution using the Shapiro-Wilk test. Measurement data that conformed to the normal distribution were expressed as median ± standard deviation (mean ± SD), and measurement data that were not normally distributed were expressed as median (interquartile range). The two groups were compared using independent sample t-test (two-tailed), non-parametric Mann-Whitney U test or Welch t-test, and all results were considered significant differences when P < 0.05. The normal reference range is the 95% normal reference range of the control group data. If the data conform to the normal distribution, the 95% normal reference range is the mean ± 1.96 times the standard deviation; if the data do not conform to the normal distribution, it is 2.5% percentile to 97.5% percentile (P0.025 ~P 0.975 ). If the value is greater than the normal reference range, it is considered positive, otherwise it is negative.

[0015] Example 1 Screening of biomarkers 1. Sample collection A total of 40 children who were admitted to our hospital for surgical treatment of CRSwNP from 2019 to 2023 were enrolled as the experimental group; 35 children who were admitted to our hospital for non-inflammatory nasal diseases such as congenital cerebrospinal fluid rhinorrhea were enrolled as the control group. Polyp tissues of localized CRSwNP, mucosal tissues of the uncinate process with normal morphology on the healthy side of localized CRSwNP, and mucosal tissues of children with non-inflammatory nasal diseases such as congenital cerebrospinal fluid rhinorrhea were collected during endoscopic sinus surgery. The separated fresh polyp tissues and mucosal tissues were quickly placed in enzyme-free EP bottles and immediately sent for RNA sequencing. The remaining tissues were immediately frozen with liquid nitrogen and stored in a -80 °C refrigerator for histopathological examination.

[0016] Demographic data collected included sex, age, allergic diseases, and number of surgeries. This study was approved by the Ethics Committee of Beijing Children's Hospital, Capital Medical University, and informed consent was obtained from all subjects (

[2022] -E-035-Y).

[0017] 2. Inclusion and Exclusion Criteria Inclusion criteria: Children who met the diagnostic criteria for pediatric CRSwNP in EPOS 2020 and had unilateral disease and underwent FESS surgery; children who were diagnosed with congenital cerebrospinal fluid rhinorrhea and choanal atresia based on clinical manifestations and imaging examinations and underwent endoscopic sinus surgery.

[0018] Exclusion criteria: children with primary ciliary dyskinesia, cystic fibrosis, fungal sinusitis, and / or recurrent lower airway infections; patients who had received antibiotics or topical / systemic steroids preoperatively.

[0019] 3. RNA sequencing: Total RNA was extracted from polyp tissue and uncinate mucosal tissue to ensure quality and library construction. After constructing qualified libraries, all samples were sequenced on the HiSeq 6000 platform (Illumina, San Diego, CA, USA). The raw reads obtained by sequencing were processed to remove low-quality sequences and joint contamination to obtain high-quality (clean) reads. All subsequent analyses were performed using clean reads. Gene expression levels were quantified as fragments per kilobase per million mapped (FPKM).

[0020] 4. Analysis of differentially expressed genes: The ANNUOYUN platform (solargenomics.com) was used to extract and visualize the results of principal component analysis (PCA) of genes. DESeq2 (RRID: SCR_015687) was used to perform differential expression analysis between the polyp group and the control group to obtain significantly differentially expressed genes (DEGs), with a cutoff value of |log2 fold-change|≥2 and a corrected p value of <0.05.

[0021] 5. Functional enrichment analysis and pathway analysis: R software (version 3.6.3; RRID: SCR_001905) was used to perform GO and KEGG pathway enrichment analysis and visualize the results. For GO and KEGG pathways, p-adjust < 0.05 was considered statistically significant and ranked in ascending order of FDR.

[0022] 6. Protein interaction network construction: DEGs were submitted to the STRING database (http: / / string-db.org / ; rid: SCR_005223) for protein–protein interaction (PPI) network construction. Cytoscape software (version 3.8.2; RRID: SCR_003032) was used. At the same time, single and non-interacting genes were removed. DEGs were analyzed using the cytoHubba plug-in in Cytoscape software (RRID: SCR_017677). The top 10 hub genes were screened using the MNC algorithm.

[0023] result: Figure 1 The GO and KEGG enrichment analysis results of DEGs in this study are shown. The results of this figure show that the biological processes are mainly related to neutrophils. The top five biological processes are neutrophil activation, neutrophil-mediated immunity, neutrophil activation involved in immune response, and neutrophil degranulation. KEGG enrichment analysis screened out a total of 18 pathways: neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, CAMP signaling pathway, IL-17 signaling pathway, etc.

[0024] We constructed a PPI network of 1419 DEGs, which contained 942 nodes and 5085 edges ( Figure 2 Left picture). We screened out 10 hub genes, namely interleukin 6 ( IL6 ), integrin subunitalpha M ( ITGAM ), CXC motif chemokine ligand 8 ( CXCL8 ), fibronectin 1 ( FN1 ), epidermal growth factor ( EGF ), integrin subunit alpha X ( ITGAX ), toll-likereceptor 8 ( TLR8 ), Fc gamma receptor IIIb ( FCGR3B ), interferon-gamma ( IFNG ), and leptin ( LEP ) ( Figure 2 right).

[0025] We used RT-qPCR to verify the expression of 10 hub genes at the RNA level ( Figure 3 Compared with the control group, FN1 ( p <0.001), CXCL8 ( p <0.01), EGF ( p <0.01)、ITGAX( p <0.05), ITGAM ( p <0.05), FCGR3B had a significant up-regulation trend, and IFNG had a significant down-regulation trend. Therefore, we further verified the protein level of these 7 genes.

[0026] We used immunohistochemical staining to detect the expression of FN1, CXCL8, EGF, ITGAX, ITGAM, FCGR3B, and IFNG at the protein level ( Figure 4 Compared with the control group, ITGAM ( p <0.0001), IFNG ( p <0.001), FCGR3B ( p <0.05), and the expression of EGF and CXCL8 showed an increasing trend in polyps.

[0027] ITGAM and FCGR3B are both localized in neutrophils, mediating immunity through neutrophil phagocytosis, and play an important role in the pathogenesis of nasal polyps. Based on the above results, we preliminarily selected ITGAM and FCGR3B as potential markers for subsequent experiments.

[0028] Example 2 Validation of ITGAM and FCGR3B in localized and recurrent CRSwNP Among the 40 children with CRSwNP, 15 were recurrent NP and 25 were localized NP. The clinical data of these children were summarized (Table 1). It was found that the serum IgE of localized NP was significantly higher than that of recurrent NP ( P <0.05).

[0029] Table 1. Clinical data of children with recurrent vs localized NP who underwent transcriptome sequencing

[0030] Data were expressed as n(%), mean ± SD, or median (interquartile range).

[0031] a. Fisher's exact test; b. Independent sample t test; c. Mann-Whitney U test; d. Welch t test Comparing the sinus CT scans of the patients with recurrence before the first surgery and at the time of recurrence, it can be seen that regardless of whether the NP was localized or diffuse before the first surgery, the NP at the time of recurrence was of maxillary sinus origin ( Figure 5 ).

[0032] With |log2 fold-change|≥2, p <0.05 was used as the cutoff value, and a total of 1133 DEGs were screened, including 532 up-regulated genes and 601 down-regulated genes. However, when |log2 fold-change|≥2 and the corrected p value <0.05 were used as the cutoff value, there were no different DEGs between localized CRSwNP and recurrent CRSwNP ( Figure 6 ). This indicates that localized CRSwNP and recurrent CRSwNP are very similar in both clinical phenotype and RNA expression levels.

[0033] Next, we used immunohistochemistry and PCR to detect ITGAM and FCGR3B in 25 localized and 15 recurrent CRSwNP polyp tissues, respectively ( Figure 7 For further evaluation ITGAM and FCGR3BThe ROC curves were drawn for the diagnostic value of PCR and immunohistochemistry results. The results of PCR showed that the AUC values ​​of ITGAM and FCGR3B in localized CRSwNP were 0.6720 and 0.6487, respectively, and in recurrent CRSwNP were 0.7004 and 0.6411, respectively. The results of immunohistochemistry showed that the AUC values ​​of ITGAM and FCGR3B in localized CRSwNP were 0.8574 and 0.7048, respectively, and in recurrent CRSwNP were 0.8241 and 0.6962, respectively. Figure 8 ).

[0034] Next, we calculated the normal reference range of immunohistochemistry. The normal reference range of ITGAM in immunohistochemistry is 0.07-0.38, and the normal reference range of FCGR3B is 0.11-0.37. A value greater than the normal reference range is considered positive, and vice versa.

[0035] Example 3 Verification of the clinical effects of ITGAM and FCGR3B as biomarkers In order to confirm the predictive ability of ITGAM and FCGR3B for CRSwNP recurrence, the research team re-enrolled 20 children who were admitted to our hospital for surgical treatment of CRSwNP in 2021. At the same time as the first surgery, immunohistochemistry detection of ITGAM and FCGR3B was performed on the polyp tissues of the children. The recurrence of the children was tracked through follow-up one year and two years after surgery. The results are shown in Table 2:

[0036]

[0037] As shown in Table 2, the recurrence rate within one year after surgery for children with positive ITGAM and / or FCGR3B tests was 71.4%, and the recurrence rate within two years after surgery was 85.7%. In addition, the recurrence rate within one year for children with double positive ITGAM and FCGR3B tests was 100%. This shows that ITGAM and / or FCGR3B have an important indicative role in the recurrence of CRSwNP, and are of great significance for postoperative medication guidance and follow-up density for children with CRSwNP.

[0038] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A biomarker for predicting the recurrence of chronic sinusitis with nasal polyps in children, characterized in that: The biomarkers are ITGAM and / or FCGR3B.

2. Use of a reagent for detecting the expression level of the biomarker according to claim 1 in the preparation of a kit for predicting the recurrence of chronic sinusitis with nasal polyps in children.

3. The use according to claim 2, characterized in that The reagents include immunohistochemical staining reagents.

4. The use according to claim 2, characterized in that The method of using the kit is: detecting the expression of ITGAM and FCGR3B in polyp tissues of children with chronic sinusitis and nasal polyps. If 1-2 of ITGAM and FCGR3B are positive, the child is judged to have a high risk of nasal polyp recurrence and needs to increase the frequency of postoperative follow-up and medication.

5. The use according to claim 4, characterized in that The normal reference range of ITGAM in immunohistochemistry is 0.07-0.38, and the normal reference range of FCGR3B is 0.11-0.

37. Values ​​greater than the normal reference range are considered positive.

6. A kit for predicting the recurrence of chronic sinusitis with nasal polyps in children, characterized in that: The kit includes reagents for detecting the expression amount of the biomarker according to claim 1.

7. The kit according to claim 6, characterized in that The reagents include immunohistochemical staining reagents.

8. The kit according to claim 6, characterized in that The method of using the kit is: detecting the expression of ITGAM and FCGR3B in polyp tissues of children with chronic sinusitis and nasal polyps. If 1-2 of ITGAM and FCGR3B are positive, the child is judged to have a high risk of nasal polyp recurrence and needs to increase the frequency of postoperative follow-up and medication.

9. The kit according to claim 8, characterized in that The normal reference range of ITGAM in immunohistochemistry is 0.07-0.38, and the normal reference range of FCGR3B is 0.11-0.

37. Values ​​greater than the normal reference range are considered positive.

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