Clinical application of shox2, ptger4 methylation combined with serum marker cyfra21-1 in diagnosis of malignant pleural mesothelioma

Abstract

The present application relates to the field of biotechnology, in particular to the clinical application of SHOX2, PTGER4 methylation and serum marker cyfra21-1 combined detection in the diagnosis of malignant pleural mesothelioma. The present application first found that the combined detection of SHOX2, PTGER4 DNA methylation and serum marker cyfra21-1 can significantly improve the diagnosis rate of malignant pleural mesothelioma, reduce misdiagnosis, improve diagnostic accuracy, and improve patient survival rate.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the clinical application of combined detection of SHOX2 and PTGER4 methylation and the serum marker cyfra21-1 in the diagnosis of malignant pleural mesothelioma. Background Technology

[0002] Mesothelioma is a rare tumor originating from mesothelial cells, which can occur in the pleura, peritoneum, pericardium, and tunica vaginalis of the testis, among other sites. Malignant pleural mesothelioma (MPM) accounts for approximately 90% of all types. The peak incidence of MPM is between 50 and 70 years of age, and it is more common in men than women. The 5-year survival rate for MPM patients is less than 10%, with a median survival of 9–12 months. MPM mainly includes epithelial and non-epithelial histological types (sarcomatoid mesothelioma, biphasic mesothelioma, and desmoplastic mesothelioma). Epithelioid mesothelioma is the most common, accounting for 70%-80% of all cases, and has the best prognosis. Studies have found a strong correlation between MPM incidence and asbestos exposure; possible pathogenic variables include mineral fibers, ionizing radiation, and simian vacuolating virus (SV40) infection. Diagnosis of MPM requires a comprehensive assessment of clinical history, imaging data, tumor markers, and histopathological morphology. Due to the lack of precise screening methods, missed diagnoses and misdiagnoses are common.

[0003] In routine clinical practice, mesothelial carcinoma (MPM) is visually difficult to distinguish from benign reactive mesothelial hyperplasia (BRT) and metastatic pleural carcinoma, posing a significant challenge to pathologists. Specifically, the high cellular morphology of MPM under a microscope, similar to that of benign reactive mesothelial hyperplasia such as tuberculosis, coupled with the common pleural effusion symptom in MPM patients, greatly confuses the diagnosis. Since MPM originates from mesothelial cells, it is crucial to differentiate it from adenocarcinoma by detecting specific proteins expressed by mesothelial and epithelial cells. MPM can be positive for at least two mesothelial markers (including Calretinin, cytokeratin 5 / 6, Wilms tumor 1, and D2-40) and negative for at least two adenocarcinoma markers (including TTF1, CEA, and BER-EP4). BAP1, an important deubiquitinating enzyme, is not expressed in MPM, especially in the epithelioid subtype, and can effectively differentiate MPM from reactive mesothelial hyperplasia, with a sensitivity of 61-67% and a specificity approaching 100%. However, because BAP1 is not expressed in all reactive mesothelial hyperplasia (MPM) diseases, setting the cutoff value is difficult and its application is limited. Furthermore, fluorescence in situ hybridization (FISH) plays an important role in the diagnosis and differential diagnosis of MPM. Chromosome 9q21 deletion is common in MPM, leading to the deletion of gene regions such as P16 (CDKN2A) and MTAP (methylthioadenosine phosphorylase). However, FISH results are inconsistent with P16 immunohistochemical staining results. The sensitivity of FISH detection for P16 deletion in MPM is 45%–85%, and false negatives are possible. Both of the above detection techniques have high specificity but low sensitivity. When BAP1 immunohistochemistry and P16 FISH are used in combination, the overall sensitivity is slightly improved, and new detection methods are urgently needed.

[0004] DNA methylation is an important DNA modification method. In MPM, asbestos can promote DNA methylation. The SFRP family (SFRP1, SFRP2, SFRP4, and SFRP5) can exert anti-cancer functions by antagonizing the Wnt signaling pathway. Hypermethylation was found in the plasma of MPM patients in 2017, but this conclusion requires further verification due to the small sample size. Furthermore, the aforementioned research group also found that the anti-cancer zinc finger protein ZIC1 is highly methylated in MPM samples, which has potential clinical diagnostic value for MPM. Currently, there are relatively few studies on MPM methylation, making its use in the diagnosis and differential diagnosis of MPM worthy of attention. Summary of the Invention

[0005] In order to overcome the problems existing in the prior art, the purpose of this invention is to provide the use of SHOX2, PTGER4 gene methylation and serum marker cyfra21-1 in combination in the preparation or screening of a reagent for detecting malignant pleural mesothelioma.

[0006] Another objective of this invention is to provide a detection kit for malignant pleural mesothelioma that uses SHOX2 and PTGER4 gene methylation and the serum marker cyfra21-1 as the detection targets.

[0007] Another objective of this invention is to provide a method for detecting malignant pleural mesothelioma using a combination of SHOX2 and PTGER4 gene methylation and the serum marker cyfra21-1 as the detection targets.

[0008] This invention is achieved through the following technical solution:

[0009] In a first aspect, the invention provides the use of the PTGER4 gene as a methylation biomarker in the preparation or screening of reagents for the detection of malignant pleural mesothelioma.

[0010] Furthermore, using the PTGER4 gene as a methylation biomarker for screening diagnostic reagents for malignant pleural mesothelioma refers to using the PTGER4 gene as a target for screening diagnostic reagents for malignant pleural mesothelioma.

[0011] Furthermore, based on the aforementioned PTGER4 gene, reagents for detecting the methylation region of the PTGER4 gene are screened for use in the preparation of a diagnostic reagent for malignant pleural mesothelioma.

[0012] Furthermore, the malignant pleural mesothelioma detection reagent contains a reagent for detecting the methylation region of the PTGER4 gene.

[0013] Furthermore, the methylated region of the PTGER4 gene is the methylated region of the PTGER4 gene promoter.

[0014] Furthermore, the reagents for detecting the methylated region of the PTGER4 gene include forward primers, reverse primers, and probes.

[0015] Furthermore, the forward primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.1, specifically: CGCGTAAGGAGTAGAAGGAG.

[0016] Furthermore, the reverse primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.2, specifically: CGTAACGATAATCACCGAACT.

[0017] Furthermore, the probe for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.3, specifically: CGATTTTTTATACGTTGGTATGTGG.

[0018] Furthermore, the malignant pleural mesothelioma detection reagent is used for the diagnosis, treatment selection, and / or prognostic assessment of malignant pleural mesothelioma.

[0019] Preferably, in a first aspect, the present invention also provides the use of the SHOX2 gene and PTGER4 gene in combination as methylation biomarkers in the preparation or screening of reagents for the detection of malignant pleural mesothelioma.

[0020] Furthermore, using the SHOX2 and PTGER4 genes together as methylation biomarkers for screening malignant pleural mesothelioma detection reagents refers to using the SHOX2 and PTGER4 genes as targets for screening malignant pleural mesothelioma detection reagents.

[0021] Furthermore, based on the aforementioned SHOX2 and PTGER4 genes, reagents for detecting the methylation region of the SHOX2 gene and the methylation region of the PTGER4 gene are screened for use in the preparation of a diagnostic reagent for malignant pleural mesothelioma.

[0022] Furthermore, the malignant pleural mesothelioma detection reagent contains reagents for detecting the methylated region of the SHOX2 gene and reagents for detecting the methylated region of the PTGER4 gene.

[0023] Furthermore, the methylated region of the SHOX2 gene is the methylated region of the SHOX2 gene promoter.

[0024] Furthermore, the methylated region of the PTGER4 gene is the methylated region of the PTGER4 gene promoter.

[0025] Furthermore, the reagents for detecting the methylated region of the PTGER4 gene include forward primers, reverse primers, and probes.

[0026] Furthermore, the forward primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.1, specifically: CGCGTAAGGAGTAGAAGGAG.

[0027] Furthermore, the reverse primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.2, specifically: CGTAACGATAATCACCGAACT.

[0028] Furthermore, the probe for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.3, specifically: CGATTTTTTATACGTTGGTATGTGG.

[0029] Furthermore, the reagents for detecting the methylated region of the SHOX2 gene include forward primers, reverse primers, and probes.

[0030] Furthermore, the forward primer for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.4, specifically: TTGTTTTTGGGTTCGGGTT.

[0031] Furthermore, the reverse primer sequence for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.5, specifically: CATAACGTAAACGCCTATACTCG.

[0032] Furthermore, the probe for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.6, specifically: ATCGAACAAACGAAACGAAAATTACC.

[0033] Furthermore, the malignant pleural mesothelioma detection reagent is used for the diagnosis, treatment selection, and / or prognostic assessment of malignant pleural mesothelioma.

[0034] Furthermore, the "combination" refers to the determination of malignant pleural mesothelioma when either the SHOX2 or PTGER4 gene in the sample is hypermethylated.

[0035] In a first aspect, the invention also provides the use of SHOX2, PTGER4 gene methylation and serum marker cyfra21-1 in combination in the preparation or screening of a reagent for detecting malignant pleural mesothelioma.

[0036] Furthermore, the combined screening of SHOX2, PTGER4 gene methylation and serum marker cyfra21-1 for malignant pleural mesothelioma detection reagents refers to using SHOX2, PTGER4 genes and cyfra21-1 as targets for screening malignant pleural mesothelioma detection reagents.

[0037] Furthermore, reagents for screening and detecting the methylation region of the SHOX2 gene and the methylation region of the PTGER4 gene based on the SHOX2 and PTGER4 genes, and reagents for screening and detecting cyfra21-1 based on the cyfra21-1 gene, are used together to prepare a reagent for detecting malignant pleural mesothelioma.

[0038] Furthermore, the malignant pleural mesothelioma detection reagent contains reagents for detecting the methylated region of the SHOX2 gene, reagents for detecting the methylated region of the PTGER4 gene, and reagents for detecting cyfra21-1.

[0039] Furthermore, the methylated region of the SHOX2 gene is the methylated region of the SHOX2 gene promoter.

[0040] Furthermore, the methylated region of the PTGER4 gene is the methylated region of the PTGER4 gene promoter.

[0041] Furthermore, the reagents for detecting the methylated region of the PTGER4 gene include forward primers, reverse primers, and probes.

[0042] Furthermore, the forward primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.1, specifically: CGCGTAAGGAGTAGAAGGAG.

[0043] Furthermore, the reverse primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.2, specifically: CGTAACGATAATCACCGAACT.

[0044] Furthermore, the probe for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.3, specifically: CGATTTTTTATACGTTGGTATGTGG.

[0045] Furthermore, the reagents for detecting the methylated region of the SHOX2 gene include forward primers, reverse primers, and probes.

[0046] Furthermore, the forward primer for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.4, specifically: TTGTTTTTGGGTTCGGGTT.

[0047] Furthermore, the reverse primer sequence for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.5, specifically: CATAACGTAAACGCCTATACTCG.

[0048] Furthermore, the probe for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.6, specifically: ATCGAACAAACGAAACGAAAATTACC.

[0049] Furthermore, the reagent for detecting cyfra21-1 includes a reagent capable of detecting cytokeratin 19 fragments in serum.

[0050] Furthermore, the reagent for detecting cyfra21-1 includes cross-linked mouse anti-human CYFRA21-1 monoclonal antibody-encoded microspheres and phycoerythrin-labeled mouse anti-human CYFRA21-1 monoclonal antibody.

[0051] Furthermore, the malignant pleural mesothelioma detection reagent is used for the diagnosis, treatment selection, and / or prognostic assessment of malignant pleural mesothelioma.

[0052] Furthermore, the combination of these three factors here refers to the diagnosis of malignant pleural mesothelioma when either SHOX2 or PTGER4 gene methylation is positive or the serum marker cyfra21-1 is highly expressed.

[0053] In a second aspect, the present invention provides the use of a PTGER4 gene methylation detection reagent in the preparation of a diagnostic kit for malignant pleural mesothelioma.

[0054] Furthermore, the malignant pleural mesothelioma detection kit contains reagents for detecting the methylation region of the PTGER4 gene.

[0055] Furthermore, the methylation region of the PTGER4 gene being detected is the methylation of the PTGER4 gene promoter region.

[0056] Furthermore, the reagents for detecting the methylated region of the PTGER4 gene include forward primers, reverse primers, and probes.

[0057] Furthermore, the forward primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.1, specifically: CGCGTAAGGAGTAGAAGGAG.

[0058] Furthermore, the reverse primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.2, specifically: CGTAACGATAATCACCGAACT.

[0059] Furthermore, the probe for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.3, specifically: CGATTTTTTATACGTTGGTATGTGG.

[0060] Furthermore, the malignant pleural mesothelioma detection kit is used for the diagnosis, treatment selection, and / or prognostic assessment of malignant pleural mesothelioma.

[0061] In a second aspect, the invention also provides the use of the SHOX2 gene methylation detection reagent and the PTGER4 gene methylation detection reagent in the preparation of a diagnostic kit for malignant pleural mesothelioma.

[0062] Furthermore, the malignant pleural mesothelioma detection kit contains reagents for detecting the methylation region of the SHOX2 gene and reagents for detecting the methylation region of the PTGER4 gene.

[0063] Furthermore, the methylated region of the SHOX2 gene is the methylated region of the SHOX2 gene promoter.

[0064] Furthermore, the methylated region of the PTGER4 gene is the methylated region of the PTGER4 gene promoter.

[0065] Furthermore, the reagents for detecting the methylated region of the PTGER4 gene include forward primers, reverse primers, and probes.

[0066] Furthermore, the forward primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.1, specifically: CGCGTAAGGAGTAGAAGGAG.

[0067] Furthermore, the reverse primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.2, specifically: CGTAACGATAATCACCGAACT.

[0068] Furthermore, the probe for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.3, specifically: CGATTTTTTATACGTTGGTATGTGG.

[0069] Furthermore, the reagents for detecting the methylated region of the SHOX2 gene include forward primers, reverse primers, and probes.

[0070] Furthermore, the forward primer for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.4, specifically: TTGTTTTTGGGTTCGGGTT.

[0071] Furthermore, the reverse primer sequence for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.5, specifically: CATAACGTAAACGCCTATACTCG.

[0072] Furthermore, the probe for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.6, specifically: ATCGAACAAACGAAACGAAAATTACC.

[0073] Furthermore, the malignant pleural mesothelioma detection kit is used for the diagnosis, treatment selection, and / or prognostic assessment of malignant pleural mesothelioma.

[0074] In a second aspect, the invention also provides the use of the SHOX2 gene methylation detection reagent, the PTGER4 gene methylation detection reagent, and the serum marker cyfra21-1 detection reagent in combination in the preparation of a diagnostic kit for malignant pleural mesothelioma.

[0075] Furthermore, the malignant pleural mesothelioma detection kit contains reagents for detecting the methylated region of the SHOX2 gene, reagents for detecting the methylated region of the PTGER4 gene, and reagents for detecting cyfra21-1.

[0076] Furthermore, the methylated region of the SHOX2 gene is the methylated region of the SHOX2 gene promoter.

[0077] Furthermore, the methylated region of the PTGER4 gene is the methylated region of the PTGER4 gene promoter.

[0078] Furthermore, the reagents for detecting the methylated region of the PTGER4 gene include forward primers, reverse primers, and probes.

[0079] Furthermore, the forward primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.1, specifically: CGCGTAAGGAGTAGAAGGAG.

[0080] Furthermore, the reverse primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.2, specifically: CGTAACGATAATCACCGAACT.

[0081] Furthermore, the probe for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.3, specifically: CGATTTTTTATACGTTGGTATGTGG.

[0082] Furthermore, the reagents for detecting the methylated region of the SHOX2 gene include forward primers, reverse primers, and probes.

[0083] Furthermore, the forward primer for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.4, specifically: TTGTTTTTGGGTTCGGGTT.

[0084] Furthermore, the reverse primer sequence for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.5, specifically: CATAACGTAAACGCCTATACTCG.

[0085] Furthermore, the probe for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.6, specifically: ATCGAACAAACGAAACGAAAATTACC.

[0086] Furthermore, the reagent for detecting cyfra21-1 includes a reagent capable of detecting cytokeratin 19 fragments in serum.

[0087] Furthermore, the reagent for detecting cyfra21-1 includes cross-linked mouse anti-human CYFRA21-1 monoclonal antibody-encoded microspheres and phycoerythrin-labeled mouse anti-human CYFRA21-1 monoclonal antibody.

[0088] Furthermore, the malignant pleural mesothelioma detection kit is used for the diagnosis, treatment selection, and / or prognostic assessment of malignant pleural mesothelioma.

[0089] A third aspect of the present invention provides a diagnostic kit for malignant pleural mesothelioma, including a PTGER4 gene methylation detection reagent.

[0090] Furthermore, the malignant pleural mesothelioma detection kit uses the PTGER4 gene as a methylation biomarker.

[0091] Furthermore, the malignant pleural mesothelioma detection kit contains a reagent for detecting methylation of the PTGER4 gene.

[0092] Furthermore, the methylation region of the PTGER4 gene being detected is the methylation of the PTGER4 gene promoter region.

[0093] Furthermore, the reagents for detecting the methylated region of the PTGER4 gene include forward primers, reverse primers, and probes.

[0094] Furthermore, the forward primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.1, specifically: CGCGTAAGGAGTAGAAGGAG.

[0095] Furthermore, the reverse primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.2, specifically: CGTAACGATAATCACCGAACT.

[0096] Furthermore, the probe for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.3, specifically: CGATTTTTTATACGTTGGTATGTGG.

[0097] Furthermore, the malignant pleural mesothelioma detection kit is used for the diagnosis, treatment selection, and / or prognostic assessment of malignant pleural mesothelioma.

[0098] A third aspect of the present invention also provides a diagnostic kit for malignant pleural mesothelioma, comprising a SHOX2 gene methylation detection reagent and a PTGER4 gene methylation detection reagent.

[0099] Furthermore, the malignant pleural mesothelioma detection kit uses the SHOX2 gene and PTGER4 gene in combination as methylation biomarkers.

[0100] Furthermore, the malignant pleural mesothelioma detection kit contains reagents for detecting the methylation region of the SHOX2 gene and reagents for detecting the methylation region of the PTGER4 gene.

[0101] Furthermore, the methylated region of the SHOX2 gene is the methylated region of the SHOX2 gene promoter.

[0102] Furthermore, the methylated region of the PTGER4 gene is the methylated region of the PTGER4 gene promoter.

[0103] Furthermore, the reagents for detecting the methylated region of the PTGER4 gene include forward primers, reverse primers, and probes.

[0104] Furthermore, the forward primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.1, specifically: CGCGTAAGGAGTAGAAGGAG.

[0105] Furthermore, the reverse primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.2, specifically: CGTAACGATAATCACCGAACT.

[0106] Furthermore, the probe for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.3, specifically: CGATTTTTTATACGTTGGTATGTGG.

[0107] Furthermore, the reagents for detecting the methylated region of the SHOX2 gene include forward primers, reverse primers, and probes.

[0108] Furthermore, the forward primer for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.4, specifically: TTGTTTTTGGGTTCGGGTT.

[0109] Furthermore, the reverse primer sequence for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.5, specifically: CATAACGTAAACGCCTATACTCG.

[0110] Furthermore, the probe for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.6, specifically: ATCGAACAAACGAAACGAAAATTACC.

[0111] Furthermore, the malignant pleural mesothelioma detection kit is used for the diagnosis, treatment selection, and / or prognostic assessment of malignant pleural mesothelioma.

[0112] A third aspect of the present invention also provides a diagnostic kit for malignant pleural mesothelioma, comprising a SHOX2 gene methylation detection reagent, a PTGER4 gene methylation detection reagent, and a serum biomarker cyfra21-1 detection reagent.

[0113] Furthermore, the malignant pleural mesothelioma detection kit uses SHOX2 and PTGER4 gene methylation and serum marker cyfra21-1 as the detection targets.

[0114] Furthermore, the malignant pleural mesothelioma detection kit contains reagents for detecting the methylated region of the SHOX2 gene, reagents for detecting the methylated region of the PTGER4 gene, and reagents for detecting cyfra21-1.

[0115] Furthermore, the methylated region of the SHOX2 gene is the methylated region of the SHOX2 gene promoter.

[0116] Furthermore, the methylated region of the PTGER4 gene is the methylated region of the PTGER4 gene promoter.

[0117] Furthermore, the reagents for detecting the methylated region of the PTGER4 gene include forward primers, reverse primers, and probes.

[0118] Furthermore, the forward primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.1, specifically: CGCGTAAGGAGTAGAAGGAG.

[0119] Furthermore, the reverse primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.2, specifically: CGTAACGATAATCACCGAACT.

[0120] Furthermore, the probe for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO.3, specifically: CGATTTTTTATACGTTGGTATGTGG.

[0121] Furthermore, the reagents for detecting the methylated region of the SHOX2 gene include forward primers, reverse primers, and probes.

[0122] Furthermore, the forward primer for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.4, specifically: TTGTTTTTGGGTTCGGGTT.

[0123] Furthermore, the reverse primer sequence for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.5, specifically: CATAACGTAAACGCCTATACTCG.

[0124] Furthermore, the probe for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO.6, specifically: ATCGAACAAACGAAACGAAAATTACC.

[0125] Furthermore, the reagent for detecting cyfra21-1 includes a reagent capable of detecting cytokeratin 19 fragments in serum.

[0126] Furthermore, the reagent for detecting cyfra21-1 includes cross-linked mouse anti-human CYFRA21-1 monoclonal antibody-encoded microspheres and phycoerythrin-labeled mouse anti-human CYFRA21-1 monoclonal antibody.

[0127] Furthermore, the malignant pleural mesothelioma detection kit is used for the diagnosis, treatment selection, and / or prognostic assessment of malignant pleural mesothelioma.

[0128] A fourth aspect of the present invention provides a method for detecting malignant pleural mesothelioma, comprising detecting the methylation level of the PTGER4 gene in a sample.

[0129] A fourth aspect of the present invention also provides a method for detecting malignant pleural mesothelioma, comprising detecting the methylation levels of the SHOX2 and PTGER4 genes in a sample.

[0130] In a fourth aspect, the present invention also provides a method for detecting malignant pleural mesothelioma, comprising detecting the methylation levels of the SHOX2 gene and the PTGER4 gene in a sample and the level of the serum biomarker cyfra21-1.

[0131] As described above, the technical solution of the present invention has the following beneficial effects:

[0132] Malignant pleural mesothelioma (MPM) is a rare and highly aggressive tumor. Traditional morphological pathology often struggles to differentiate MPM from reactive mesothelial hyperplasia. This invention, for the first time, explores the clinical value of SHOX2 and PTGER4 gene methylation detection and the serum biomarker cyfra21-1 in the diagnosis of MPM. Results show that combined detection of SHOX2 and PTGER4 DNA methylation and the serum biomarker cyfra21-1 significantly improves the diagnostic rate of MPM, reduces misdiagnosis, increases diagnostic accuracy, and improves patient survival. Attached Figure Description

[0133] Figure 1 HE-stained images of MPM and reactive mesothelioma controls, where A: HE-stained image of MPM; B: HE-stained image of reactive mesothelioma controls. Scale bar = 50 μm; MPM, malignant pleural mesothelioma.

[0134] Figure 2 ROC curve analysis was used to analyze the diagnostic value of various methylation indicators and tumor markers. Among them, A: ROC curve analysis of the diagnostic value of SHOX2, PTGER4, RASSF1A, SEPTIN9 and HOXA9 gene methylation; B: ROC curve analysis of the diagnostic value of blood CEA, NSE, Pro-GRP, SCC, CYFRA21-1, AFP, CA-125, CA15-3 and CA19-9.

[0135] Figure 3 ROC curves show methylation indicators and tumor markers with good diagnostic efficacy. Specifically: A: ROC curves show the diagnostic value of SHOX2, PTGER4, and HOXA9 gene methylation. B: ROC curves show the diagnostic value of cyfra21-1. C: ROC curves compare the diagnostic value of PTGER4 combined with SHOX2, PTGER4 combined with HOXA9, PTGER4 combined with RASSF1A, and PTGER4 combined with SEPTIN9 methylation. D: ROC curves show the diagnostic value of SHOX2 and PTGER4 methylation combined with the tumor marker cyfra21-1.

[0136] Figure 4 Distribution of SHOX2 and PTGER4 methylation ΔCt and cyfra21-1 in benign and malignant mesothelioma lesions. A: Distribution of SHOX2 methylation ΔCt values ​​in benign and malignant mesotheliomas; B: Distribution of PTGER4 methylation ΔCt values ​​in benign and malignant mesotheliomas; C: Distribution of Cyfra21-1 in benign and malignant mesotheliomas. "ΔCt = 20" indicates no fluorescence signal detected, a negative result. MPM, malignant pleural mesothelioma.

[0137] Figure 5 The relationship between SHOX2, PTGER4 methylation and cyfra21-1 and overall survival in MPM patients, where A: the relationship between SHOX2 methylation and overall survival in MPM patients; B: the relationship between PTGER4 methylation and overall survival in MPM patients; and C: the relationship between cyfra21-1 and overall survival in MPM patients. Detailed Implementation

[0138] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the process equipment or apparatus not specifically specified in the following embodiments are all conventional equipment or apparatus in the art. Furthermore, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated; it should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in the present invention does not exclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned two devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is only a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0139] Example 1

[0140] 1. Materials and Methods

[0141] 1.1 Patients

[0142] Forty-epithelial / cytological paraffin specimens from 48 cases of mesothelial hyperplasia (MPM) and 42 cases of benign reactive mesothelial hyperplasia were selected from the pathology department of a hospital in Beijing between May 2018 and May 2022. Two MPM patients had both histological and cytological paraffin specimens. There were 53 males and 35 females, aged 14–84 years, with a median age of 58.6 years. 33 patients had a history of smoking, and 55 had no smoking history. All cases were independently evaluated and diagnosed by two qualified pathologists, who also assessed and recorded the mesothelial cell count. Patients with more than 10% mesothelial cells were included in the study cohort. The research involved in this invention was approved by the hospital's human research ethics committee.

[0143] 1.2 Nucleic acid extraction and DNA methylation detection

[0144] Depending on tissue size, collect approximately 3–5 paraffin sections in enzyme-free 1.5 mL centrifuge tubes. Genomic DNA was extracted using a standard kit (AmoyDx, Xiamen, China). 4.0 fluorometer (Life Technologies, USA) was used to assess the concentration, and DNA samples were normalized to 10 ng / μL. If the concentration was too low, the sample should be re-extracted or excluded. Methylation modification step: 200 ng of DNA was treated with sodium bisulfite using a commercial DNA purification kit (PF03X056, Shanghai TransGen Biotech Co., Ltd., China). All bisulfite-treated DNA was purified and detected using a SLAN 96SReal-Time PCR system (Shanghai, China). The reaction parameters were set to 98°C for 8 minutes, 64°C for 3.5 hours, and stored at 4°C. After purification of the bisulfite-modified DNA, methylation-specific fragments were amplified. The ACTB gene was used as an internal control. DNA polymerase:PCR reaction mixture = 15:0.3. Each assay system contained 15 μL of PCR reaction solution and 5 μL of DNA sample. PCR amplification and signal acquisition steps: Stage 1: 95℃ for 10 min, 1 cycle; Stage 2: 95℃ for 15 s, 60℃ for 30 s, 5 cycles; Stage 3: 95℃ for 15 s, 57℃ for 30 s, a total of 40 cycles (acquiring FAM, VIC / HEX, and CY5 signals). The degree of methylation is assessed using the following formulas: the smaller the ΔCt, the stronger the methylation signal. ΔCtSHOX2 = CtSHOX2 - CtACTB, ΔCtPTGER4 = CtPTGER4 - CtACTB. When the fluorescence signal amplification curve is a smooth "S" shape and the Ct value of ACTB is between 18 and 27, the result is considered valid. Plot the receiver operating characteristic (ROC) curve and calculate the Youden index: sensitivity - (1 - specificity) to obtain the optimal cutoff value. A positive result for either the SHOX2 or PTGER4 gene indicates a positive methylation detection result; otherwise, the result is negative.

[0145] Exemplarily, the reagents used in this embodiment of the invention for detecting the methylated region of the PTGER4 gene include a forward primer, a reverse primer, and a probe. The forward primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO. 1, specifically: CGCGTAAGGAGTAGAAGGAG. The reverse primer for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO. 2, specifically: CGTAACGATAATCACCGAACT. The probe for detecting the methylated region of the PTGER4 gene contains the sequence shown in SEQ ID NO. 3, specifically: CGATTTTTTATACGTTGGTATGTGG.

[0146] Exemplarily, the reagents used in this embodiment of the invention for detecting the methylated region of the SHOX2 gene include a forward primer, a reverse primer, and a probe. The forward primer for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO. 4, specifically: TTGTTTTTGGGTTCGGGTT. The reverse primer for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO. 5, specifically: CATAACGTAAACGCCTATACTCG. The probe for detecting the methylated region of the SHOX2 gene contains the sequence shown in SEQ ID NO. 6, specifically: ATCGAACAAACGAAACGAAAATTACC.

[0147] For example, the reagents used in the embodiments of the present invention for detecting cyfra21-1 are selected from reagents that can detect cytokeratin 19 fragments in serum, including microspheres encoded by cross-linked mouse anti-human CYFRA21-1 monoclonal antibody and phycoerythrin-labeled mouse anti-human CYFRA21-1 monoclonal antibody.

[0148] 1.3 Follow-up

[0149] Eighty-eight patients were followed up by telephone from the time of pathological diagnosis until their death or April 20, 2023. Survival time was measured in months, with the longest follow-up time being 59 months and the median follow-up time being 20 months. Two patients were lost to follow-up, 34 died, and 54 survived.

[0150] 1.4 Statistical Analysis

[0151] Data were statistically analyzed using SPSS 21.0 (SPSS, Chicago, IL) software. Quantitative data were expressed as mean ± standard deviation (SD). Independent samples t-tests were used for comparisons between groups. The relationship between methylation levels and clinicopathological features was analyzed using the χ² test or Fisher's exact test. Kaplan-Meier survival analysis was performed. A p-value < 0.05 was considered statistically significant, and the significance level was set at α = 0.05 (two-tailed).

[0152] 2 Results

[0153] 2.1 Clinical and pathological data

[0154] The clinicopathological data of the 88 patients included in the study are shown in Table 1. Both groups of patients presented with varying degrees of symptoms including chest tightness, shortness of breath, fever, fatigue, cough, sputum production, chest pain, wheezing, and hemoptysis. The top five most common symptoms in the MPM group and the benign control group are shown in Table 1. Notably, the vast majority of patients had pleural effusion. 35 patients (76.1%) in the MPM group had pleural effusion symptoms. Routine morphology, especially interstitial involvement, remains the most helpful method for differentiating between benign and malignant mesothelial diseases. However, in some cases, typical functional features are not available, such as… Figure 1 As shown, similar staining patterns and cellular components to MPM and reactive mesothelial hyperplasia, it is difficult to differentiate MPM from reactive mesothelial hyperplasia based solely on conventional pathological morphology, leading to a high probability of misdiagnosis. In fact, 14 MPM patients were misdiagnosed with tuberculosis and received anti-tuberculosis drug treatment for up to 1.5 years. This demonstrates that MPM is easily mistaken for tuberculosis, thus delaying the ideal time for patient treatment.

[0155] Table 1. Patient's clinicopathological data

[0156]

[0157]

[0158] 2.2 ROC analysis of the diagnostic efficacy of SHOX2, PTGER4 methylation and serum marker cyfra21-1 levels for MPM

[0159] In the initial screening, nine methylation markers (APC, ESR1, RASSF1A, SHOX2, PTGER4, CDH1, CDKN2A, SEPTIN9, and HOXA9) were selected from paraffin samples for the diagnosis of MPM. Markers with relatively poor specificity, namely APC, CDH1, CDKN2A, and ESR1 (data not shown), were excluded. ROC curve analysis was then used to assess the diagnostic efficacy of five methylation markers (RASSF1A, SHOX2, PTGER4, SEPTIN9, and HOXA9) and nine serum markers (CEA, NSE, Pro-GRP, SCC, cyfra21-1, AFP, CA-125, CA-153, and CA-199) in paraffin samples. Figure 2 ).like Figure 3 As shown in Figure A, the methylation levels of SHOX2 (0.817), PTGER4 (0.841), and HOXA9 (0.696) were all higher than other methylation markers, indicating that these biomarkers have high diagnostic efficacy for MPM. Since HOXA9 methylation-positive cases were covered by PTGER4 methylation, the AUCs for diagnosing MPM with PTGER4 combined with SHOX2, PTGER4 combined with HOXA9, PTGER4 combined with RASSF1A, and PTGER4 combined with SEPTIN9 were 0.927, 0.849, 0.829, and 0.838, respectively. Figure 3 Therefore, SHOX2 combined with PTGER4 methylation was chosen as the combined detection indicator. The AUC of the serum biomarker cyfra21-1 (0.872) was significantly higher than that of other serum biomarkers (C). Figure 2 B), the AUC of the combined detection of SHOX2, PTGER4 and cyfra21-1 was 0.980 ( Figure 3 D) indicates that the combined detection of the three indicators has good diagnostic value for MPM.

[0160] 2.3 Sensitivity and specificity of SHOX2, PTGER4 methylation and serum biomarker cyfra21-1 in diagnosing MPM

[0161] Scatter plot Figure 4 This displays the levels of SHOX2, PTGER4 methylation ΔCt, and cyfra21-1 in all MPM patients and patients with benign reactive mesothelial hyperplasia. (See also:) Figure 4As shown, SHOX2, HOXA9, and PTGER4 methylation and the serum biomarker cyfra21-1 were significantly higher in the MPM group than in the benign control group (P<0.05). According to the Youden index formula, the ideal ΔCt cutoff values ​​for SHOX2 and PTGER4 methylation were 9 and 7, respectively, to obtain higher sensitivity and specificity. The cutoff value (6 U / ml) of the serum biomarker cyfra21-1 was determined according to the manufacturer's instructions and confirmed by scatter plotting. Figure 4 ).

[0162] The sensitivities of SHOX2, PTGER4 and cyfra21-1 methylation for detecting MPM were 43.5% (20 / 46), 58.7% (27 / 46) and 47.8% (22 / 46), respectively, and the specificities were 100% (42 / 42), 97.6% (41 / 42) and 100.0% (42 / 42), respectively. The combined sensitivity of SHOX2 and PTGER4 in diagnosing MPM was 87.0% (40 / 46), and the specificity was 97.6% (41 / 42). The combined sensitivity of PTGER4 and HOXA9 was 60.9% (28 / 46), and the specificity was 95.2% (40 / 42). The combined sensitivity of PTGER4 and RASSF1A was 63.0% (29 / 46), and the specificity was 97.6% (41 / 42). The combined sensitivity of PTGER4 and SEPTIN9 was also 60.9% (28 / 46), and the specificity was 97.6% (41 / 42). The combined sensitivity of SHOX2 and PTGER4 was significantly higher than other combinations, while the combined sensitivity of SHOX2, PTGER4, and cyfra21-1 in diagnosing MPM was 95.7% (44 / 46), and the specificity was 97.6% (41 / 42) (Table 2). The "combination" here refers to a diagnosis of malignant pleural mesothelioma when either SHOX2 or PTGER4 gene methylation is positive in the sample, or when the serum marker cyfra21-1 is highly expressed. Further studies found no statistically significant association between SHOX2, PTGER4, and cyfra21-1 methylation levels and MPM patients' sex, age, smoking history, asbestos exposure history, or histological subtype. This suggests that methylation detection combined with serum markers can be used for the diagnosis of MPM and benign reactive mesothelial hyperplasia.

[0163] Table 2. Sensitivity and specificity of SHOX2, PTGER4 methylation and serum marker cyfra21-1 in diagnosing MPM.

[0164]

[0165]

[0166] Note: CI, confidence interval; MPM: malignant mesothelioma

[0167] 2.4 Correlation between SHOX2, PTGER4 methylation and cyfra21-1 levels and patient prognosis

[0168] Kaplan-Meier survival analysis showed the relationship between SHOX2 and PTGER4 methylation, serum marker cyfra21-1 levels, and overall patient survival. Figure 5 High levels of cyfra21-1 predicted a poor prognosis in MPM patients (p = 0.003). PTGER4 hypermethylation may also indicate a poor prognosis, but the difference was not statistically significant (p > 0.05). SHOX2 hypermethylation was not significantly associated with MPM patient prognosis (p > 0.05). In the MPM case group, the median survival time for SHOX2-positive and negative patients was 17 months and 18 months, respectively, and the median survival time for PTGER4-positive and negative patients was 17 months and 18 months, respectively, with no statistically significant difference, possibly due to the small number of cases. The median survival time for patients with high and low cyfra21-1 expression was 12 months and 22 months, respectively, showing a significant difference.

[0169] 3 Discussion

[0170] MPM is a highly malignant tumor with a poor prognosis, closely associated with a history of asbestos exposure, typically occurring 20–40 years after exposure. Data shows that in 2020, there were 30,870 new cases of MPM globally, accounting for 0.2% of all new malignant tumors, and 26,278 deaths, representing 0.3% of all malignant tumor deaths worldwide. Although asbestos has been banned in many countries, its negative impacts persist.

[0171] MPM originates from mesothelial cells and is morphologically difficult to distinguish from reactive mesothelial proliferative diseases, especially in patients with pleural effusion. Due to a lack of crucial information about tissue structure and growth patterns, differentiating its exact nature by observing the shape of exfoliated cells is a significant challenge. Accurate diagnosis of benign and malignant MPM is crucial for patient treatment and prognostic assessment. Commonly used pathological techniques such as immunohistochemistry, fluorescence in situ hybridization, and sequencing each have their advantages and disadvantages. Compared to other tumors, methylation detection techniques are rarely used for the differential diagnosis of MPM and mesothelial proliferative diseases. In our cohort, the methylation levels of SHOX2 and PTGER4 in MPM tissues / cells were higher than in benign controls. According to ROC curves, the AUC values ​​for SHOX2 and PTGER4 were 0.817 and 0.841, respectively, while the AUC value for the blood tumor marker cyfra21-1 was 0.872. The AUC value of SHOX2 combined with PTGER4 was 0.927, significantly higher than other methylation combinations, while the diagnostic efficacy of SHOX2, PTGER4, and the serum biomarker cyfra21-1 combined was as high as 0.980. In MPM, the diagnostic sensitivity and specificity of SHOX2 were 43.5% and 97.6%, respectively; those of PTGER4 were 58.7% and 100%, respectively; and those of cyfra21-1 were 45.7% and 100%, respectively. The diagnostic sensitivity of SHOX2 combined with PTGER4 was 87.0% (40 / 46), and the specificity was 97.6% (41 / 42). The sensitivity and specificity of the three-indicator combination were 95.7% and 97.6%, respectively, indicating that the three-indicator combined methylation can effectively differentiate MPM from benign mesothelial hyperplasia, with PTGER4 showing better diagnostic efficacy. To date, no association has been found between PTGER4 and MPM. This invention performed Kaplan-Meier survival analysis on all included cases and found that patients with high cyfra21-1 expression had significantly shorter survival times than those with low expression. However, in the MPM case group, there was no significant difference in survival between SHOX2 and PTGER4 expression, which may be related to the limited number of cases.

[0172] This invention includes patients with MPM and reactive mesothelial hyperplasia, whose pathological diagnoses are easily confused. Methylation of the SHOX2 and PTGER4 genes and the serum marker cyfra21-1 levels were detected to explore the clinical application value of their combined detection in the differential diagnosis of MPM.

[0173] In conclusion, this study demonstrates that the combined detection of SHOX2 and PTGER4 gene methylation and the serum biomarker cyfra21-1 has significant value in differentiating between mesothelial cell proliferative disorder (MPM) and reactive mesothelial cell hyperplasia. Incorporating methylation detection results into the analysis of clinically suspected MPM lesions can improve the detection rate of MPM. As an effective diagnostic tool supplementing traditional pathological diagnosis, it can assist pathologists in making a definitive diagnosis, contributing to the early diagnosis and treatment of the disease.

[0174] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. Application of detection reagents targeting PTGER4 gene methylation biomarkers in the preparation of diagnostic reagents for malignant pleural mesothelioma.

2. The use according to claim 1, characterized in that, The diagnostic reagent for malignant pleural mesothelioma contains a reagent for detecting the methylation region of the PTGER4 gene.

3. The use according to claim 2, characterized in that, It also includes one or more of the following features: the reagent for detecting the methylated region of the PTGER4 gene includes a forward primer, a reverse primer, and a probe; the sequence of the forward primer for detecting the methylated region of the PTGER4 gene is shown in SEQ ID NO.1; the sequence of the reverse primer for detecting the methylated region of the PTGER4 gene is shown in SEQ ID NO.2; The sequence of the probe used to detect the methylated region of the PTGER4 gene is shown in SEQ ID NO.

3.

4. The use of a combination of a detection reagent for SHOX2 gene methylation biomarkers and a detection reagent for PTGER4 gene methylation biomarkers in the preparation of a diagnostic reagent for malignant pleural mesothelioma, wherein the combination refers to the determination of malignant pleural mesothelioma when either SHOX2 or PTGER4 gene is highly methylated in the sample.

5. The use according to claim 4, characterized in that, The diagnostic reagent for malignant pleural mesothelioma contains reagents for detecting the methylation region of the SHOX2 gene and reagents for detecting the methylation region of the PTGER4 gene.

6. The use according to claim 5, characterized in that, It also includes one or more of the following features: the reagent for detecting the methylated region of the PTGER4 gene includes a forward primer, a reverse primer, and a probe; the reagent for detecting the methylated region of the SHOX2 gene includes a forward primer, a reverse primer, and a probe.

7. The use according to claim 6, characterized in that, It also includes any one or more of the following features: the sequence of the forward primer for detecting the methylated region of the PTGER4 gene is shown in SEQ ID NO.1; the sequence of the reverse primer for detecting the methylated region of the PTGER4 gene is shown in SEQ ID NO.2; the sequence of the probe for detecting the methylated region of the PTGER4 gene is shown in SEQ ID NO.3; the sequence of the forward primer for detecting the methylated region of the SHOX2 gene is shown in SEQ ID NO.4; the sequence of the reverse primer for detecting the methylated region of the SHOX2 gene is shown in SEQ ID NO.5; and the sequence of the probe for detecting the methylated region of the SHOX2 gene is shown in SEQ ID NO.

6.

8. The use of a combination of a detection reagent for SHOX2 gene methylation biomarker, a detection reagent for PTGER4 gene methylation biomarker, and a detection reagent for serum biomarker cyfra21-1 in the preparation of a diagnostic reagent for malignant pleural mesothelioma, wherein the combination refers to the diagnosis of malignant pleural mesothelioma when either SHOX2 or PTGER4 gene methylation is positive or serum biomarker cyfra21-1 is highly expressed.

9. The use according to claim 8, characterized in that, The diagnostic reagent for malignant pleural mesothelioma contains reagents for detecting the methylation region of the SHOX2 gene, reagents for detecting the methylation region of the PTGER4 gene, and reagents for detecting cyfra21-1.

10. The use according to claim 9, characterized in that, It also includes one or more of the following features: the reagent for detecting the methylated region of the PTGER4 gene includes a forward primer, a reverse primer, and a probe; the reagent for detecting the methylated region of the SHOX2 gene includes a forward primer, a reverse primer, and a probe; the reagent for detecting cyfra21-1 includes a reagent capable of detecting cytokeratin 19 fragments in serum.

11. The use according to claim 10, characterized in that, It also includes any one or more of the following features: the sequence of the forward primer for detecting the methylated region of the PTGER4 gene is shown in SEQ ID NO.1; the sequence of the reverse primer for detecting the methylated region of the PTGER4 gene is shown in SEQ ID NO.2; the sequence of the probe for detecting the methylated region of the PTGER4 gene is shown in SEQ ID NO.3; the sequence of the forward primer for detecting the methylated region of the SHOX2 gene is shown in SEQ ID NO.4; the sequence of the reverse primer for detecting the methylated region of the SHOX2 gene is shown in SEQ ID NO.5; and the sequence of the probe for detecting the methylated region of the SHOX2 gene is shown in SEQ ID NO.

6. The reagents for detecting cyfra21-1 include: cross-linked mouse anti-human CYFRA21-1 monoclonal antibody-encoded microspheres and phycoerythrin-labeled mouse anti-human CYFRA21-1 monoclonal antibody.

12. Application of PTGER4 gene methylation detection reagent in the preparation of diagnostic kits for malignant pleural mesothelioma.

13. The use according to claim 12, characterized in that, The diagnostic kit for malignant pleural mesothelioma contains reagents for detecting the methylation region of the PTGER4 gene.

14. The use according to claim 13, characterized in that, The reagent for detecting the methylated region of the PTGER4 gene includes a forward primer, a reverse primer, and a probe; the sequence of the forward primer for detecting the methylated region of the PTGER4 gene is shown in SEQ ID NO.1; the sequence of the reverse primer for detecting the methylated region of the PTGER4 gene is shown in SEQ ID NO.

2. The sequence of the probe used to detect the methylated region of the PTGER4 gene is shown in SEQ ID NO.

3.

15. Application of SHOX2 gene methylation detection reagent and PTGER4 gene methylation detection reagent in the preparation of diagnostic kits for malignant pleural mesothelioma.

16. The use according to claim 15, characterized in that, The diagnostic kit for malignant pleural mesothelioma contains reagents for detecting the methylation region of the SHOX2 gene and reagents for detecting the methylation region of the PTGER4 gene.

17. The use according to claim 16, characterized in that, The reagent for detecting the methylated region of the PTGER4 gene includes a forward primer, a reverse primer, and a probe; the reagent for detecting the methylated region of the SHOX2 gene includes a forward primer, a reverse primer, and a probe; the sequence of the forward primer for detecting the methylated region of the PTGER4 gene is shown in SEQ ID NO.1; the sequence of the reverse primer for detecting the methylated region of the PTGER4 gene is shown in SEQ ID NO.2; the sequence of the probe for detecting the methylated region of the PTGER4 gene is shown in SEQ ID NO.3; the sequence of the forward primer for detecting the methylated region of the SHOX2 gene is shown in SEQ ID NO.4; the sequence of the reverse primer for detecting the methylated region of the SHOX2 gene is shown in SEQ ID NO.5; and the sequence of the probe for detecting the methylated region of the SHOX2 gene is shown in SEQ ID NO.

6.

18. The use of SHOX2 gene methylation detection reagent, PTGER4 gene methylation detection reagent and serum marker cyfra21-1 detection reagent in the preparation of a diagnostic kit for malignant pleural mesothelioma.

19. The use according to claim 18, characterized in that, The diagnostic kit for malignant pleural mesothelioma contains reagents for detecting the methylation region of the SHOX2 gene, reagents for detecting the methylation region of the PTGER4 gene, and reagents for detecting cyfra21-1.

20. The use according to claim 19, characterized in that, The reagent for detecting the methylation region of the PTGER4 gene includes a forward primer, a reverse primer, and a probe; the reagent for detecting the methylation region of the SHOX2 gene includes a forward primer, a reverse primer, and a probe; the reagent for detecting cyfra21-1 includes a reagent capable of detecting cytokeratin 19 fragments in serum; the sequence of the forward primer for detecting the methylation region of the PTGER4 gene is shown in SEQ ID NO.1; the sequence of the reverse primer for detecting the methylation region of the PTGER4 gene is shown in SEQ ID NO.2; the sequence of the probe for detecting the methylation region of the PTGER4 gene is shown in SEQ ID NO.3; the sequence of the forward primer for detecting the methylation region of the SHOX2 gene is shown in SEQ ID NO.4; the sequence of the reverse primer for detecting the methylation region of the SHOX2 gene is shown in SEQ ID NO.5; the sequence of the probe for detecting the methylation region of the SHOX2 gene is shown in SEQ ID NO.

4. As shown in NO.6; the reagent for detecting cyfra21-1 includes cross-linked mouse anti-human CYFRA21-1 monoclonal antibody-encoded microspheres and phycoerythrin-labeled mouse anti-human CYFRA21-1 monoclonal antibody.

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