Application of reagent for detecting phosphatidylinositol proteoglycan 3 in preparation of product for diagnosing pleural pulmonary blastoma

Abstract

The invention provides application of a reagent for detecting phosphatidylinositol proteoglycan 3 in preparation of a product for diagnosing pleural pulmonary blastoma, and relates to the technical field of tumors. The research of the inventor finds that compared with healthy tissues or congenital cystic pulmonary disease tissues, the differential expression of the phosphatidylinositol proteoglycan 3 in pleural pulmonary blastoma tissues is up-regulated, and the expression level of the phosphatidylinositol proteoglycan 3 is related to the prognosis of pleural pulmonary blastoma, so that the phosphatidylinositol proteoglycan 3 can be used for treating the pleural pulmonary blastoma The phosphatidylinositol proteoglycan 3 can be used as a marker for diagnosis of pleural pulmonary blastoma, and has good sensitivity and specificity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumors, in particular to the application of a reagent for detecting glypican 3 in the preparation of a product for diagnosing pleuropulmonary blastoma. BACKGROUND

[0002] 1.1 Introduction to the background of pleuropulmonary blastoma disease Pleuropulmonary blastoma (PPB) is a rare primary lung malignant mesenchymal tumor in children, which is highly invasive, lacks specific clinical manifestations, is easily misdiagnosed, and has a high mortality rate. The etiology and pathogenesis of PPB are still unknown. PPB has special pathological features, mainly composed of primitive germinal and a malignant mesenchymal stromal cell, and generally originates from the pleura, lung or mediastinum. Some originate from lung damage caused by congenital pulmonary cystic variation, and have a family incidence tendency. Studies have shown that DICER1 gene mutation is the mechanism of family incidence.

[0003] PPB is rare, accounting for 0.25%~0.5% of primary lung malignant tumors. The incidence of PPB is about 1 / 250 000, of which 93% occurs in children under the age of 6 and infants, accounting for about 2.3% of lung lesions in children. The International Pleuropulmonary Blastoma Registry has reported about 350 cases of childhood PPB disease. Clinically, PPB is mainly divided into three types: type I (cystic), type II (cystic-solid), and type III (solid). There are differences in the age of onset and prognosis of children with different types of PPB. Professor Zai et al. reported 22 cases of PPB disease in China. The prognosis of type I PPB is good, but the 2-year survival rate of type II and type III PPB is only 27.13%, and the 5-year survival rate is as low as 9.11%. Type II and type III are prone to recurrence and distant metastasis, and the prognosis is extremely poor. Studies have shown that type I PPB can progress to type II and type III, and this pathological transformation is an important factor leading to tumor-related death in children with type I PPB. If early type I PPB is not diagnosed and treated in time, tumor cells may rapidly metastasize to the brain, bones, liver, pancreas, kidneys, and adrenal glands, missing the best treatment opportunity. Therefore, early diagnosis and treatment of PPB will have important positive significance for improving the quality of life of children with PPB.

[0004] 1.2 Lack of reliable pathological diagnostic markers for childhood pleuropulmonary blastoma PPB is a heterogeneous malignancy with some pathological features of other childhood tumors such as rhabdomyosarcoma, Wilms' tumor, and malignant germ cell tumor. PPB can originate from the lung itself, the pleura and mediastinum, and also from its own cystic lung lesions, mainly composed of primitive blastemic and malignant mesenchymal stromal cells, which can differentiate into rhabdomyosarcoma, chondrosarcoma or liposarcoma. In the early stage of the tumor, there is a transition between the normally developing lung tissue and the tumor, which is manifested as the proliferation of mesenchymal cells in the alveolar septum, leading to its uniform expansion and thickening, that is, the histological change of type I PPB. With the progression of the tumor, the proliferating mesenchymal cells form solid nodules that protrude into the cystic cavity, which is generally manifested as a cystic-solid structure, that is, type II PPB; finally, the solid nodules completely replace the cystic area, that is, type III PPB. During this transformation process, the proliferating mesenchymal cells can differentiate into rhabdomyoblasts, cartilage nodules, etc., and the cell atypia gradually becomes obvious. Most tumor cells can be seen positive expression of vimentin staining, and the respiratory epithelial cells covering the cystic cavity have positive expression of cytokeratin (CK) staining in the gas-filled cavities of the tumor solid area. Rhabdomyoblasts and sarcomas can express specific muscle fiber proteins (muscle specific actin, MSA) and desmin, etc. The results of immunohistochemical staining of the above-mentioned various markers can be used as an important reference for the pathological diagnosis of PPB, but so far there is still a lack of a reliable pathological marker for evaluating the prognosis of PPB. Early diagnosis of PPB and timely adoption of effective treatment measures are of great significance to the survival prognosis of the children. Early diagnosis, early surgical resection and appropriate chemotherapy or radiotherapy of PPB are the key factors to improve the clinical outcome. Due to the pathological characteristics of high invasiveness and easy metastasis of PPB, it often lacks specific clinical manifestations and is easily confused with respiratory tract infections and other diseases, and is easily misdiagnosed. Children with PPB often seek medical treatment due to respiratory symptoms, such as cough, sputum, chest tightness and shortness of breath, chest pain or fever of unknown origin, which are non-specific clinical manifestations and are easy to be misdiagnosed or missed. Type I PPB is often diagnosed as congenital pulmonary airway malformation (CPAM), which is another clinically common non-tumorous cystic lung disease. The diagnosis of PPB in clinic is mainly based on the comprehensive judgment of clinical manifestations, pathology, cytogenetics and imaging examination, and there is still a lack of reliable pathological diagnostic markers.

[0005] Therefore, the present application is provided. SUMMARY

[0006] The first object of the present application is to provide the use of a reagent for detecting glypican 3 (GPC3) in the preparation of a product for diagnosing pleuropulmonary blastoma, so as to solve the above technical problems.

[0007] A second object of the present invention is to provide the use of a reagent for detecting phosphatidylinositol proteoglycan 3 in the preparation of products for assessing the prognosis of pleural pulmonary blastoma.

[0008] A third objective of this invention is to provide a kit for diagnosing or assessing the prognosis of pleural pulmonary blastoma.

[0009] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides the application of a reagent for detecting phosphatidylinositol proteoglycan 3 in the preparation of products for diagnosing pleural pulmonary blastoma.

[0010] As a further technical solution, the samples tested by the reagent include lung tissue and blood.

[0011] As a further technical solution, the expression of phosphatidylinositol proteoglycan 3 is upregulated in pleural pulmonary blastoma.

[0012] As a further technical solution, the product includes a reagent kit.

[0013] Secondly, the present invention provides the use of reagents for detecting phosphatidylinositol proteoglycan 3 in the preparation of products for assessing the prognosis of pleural pulmonary blastoma.

[0014] As a further technical solution, the samples tested by the reagent include lung tissue and blood.

[0015] As a further technical solution, the expression of phosphatidylinositol proteoglycan 3 is upregulated in pleural pulmonary blastoma.

[0016] As a further technical solution, the product includes a reagent kit.

[0017] Thirdly, the present invention provides a kit for diagnosing pleural pulmonary blastoma or assessing the prognosis of pleural pulmonary blastoma, the kit being based on immunohistochemical staining to detect the expression level of phosphatidylinositol proteoglycan 3, comprising a primary antibody, a horseradish peroxidase-labeled secondary antibody, and a dye. The primary antibody includes an antiphosphatidylinositol proteoglycan 3 antibody; The secondary antibody specifically recognizes the primary antibody.

[0018] As a further technical solution, the dye includes hematoxylin.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The inventors discovered that, compared to healthy tissue or congenital cystic lung lesions, phosphatidylinositol proteoglycan 3 is differentially upregulated in pleural pulmonary blastoma tissue, and its expression level is related to the prognosis of pleural pulmonary blastoma. Therefore, phosphatidylinositol proteoglycan 3 can be used as a marker for the diagnosis of pleural pulmonary blastoma with good sensitivity and specificity. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 The staining results for PPB, adjacent normal tissue, and congenital cystic lung lesions; Figure 2 To investigate the relationship between differential GPC3 expression in pleural pulmonary blastoma and survival prognosis. Detailed Implementation

[0022] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0023] In a first aspect, the present invention provides the application of a reagent for detecting phosphatidylinositol proteoglycan 3 in the preparation of products for diagnosing pleural pulmonary blastoma.

[0024] The inventors discovered that, compared to healthy tissue or congenital cystic lung lesions, phosphatidylinositol proteoglycan 3 is differentially upregulated in pleural pulmonary blastoma tissue. Therefore, phosphatidylinositol proteoglycan 3 can be used as a biomarker for the diagnosis of pleural pulmonary blastoma with good sensitivity and specificity.

[0025] In some alternative implementations, the samples tested by the reagent include lung tissue and blood.

[0026] In some alternative embodiments, the product includes a kit. This kit is capable of detecting phosphatidylinositol proteoglycan 3.

[0027] Secondly, the present invention provides the use of reagents for detecting phosphatidylinositol proteoglycan 3 in the preparation of products for assessing the prognosis of pleural pulmonary blastoma.

[0028] The inventors discovered that, compared to healthy tissue or congenital cystic lung lesions, phosphatidylinositol proteoglycan 3 is differentially upregulated in pleural pulmonary blastoma tissue, and its expression level is related to the prognosis of pleural pulmonary blastoma. Therefore, phosphatidylinositol proteoglycan 3 can be used as a biomarker to assess the prognosis of pleural pulmonary blastoma, and it has good sensitivity and specificity.

[0029] In some alternative implementations, the samples tested by the reagent include lung tissue and blood.

[0030] In some alternative embodiments, the phosphatidylinositol proteoglycan 3 is upregulated in pleural pulmonary blastoma.

[0031] Thirdly, the present invention provides a kit for diagnosing pleural pulmonary blastoma or assessing the prognosis of pleural pulmonary blastoma, the kit being based on immunohistochemical staining to detect the expression level of phosphatidylinositol proteoglycan 3, comprising a primary antibody, a horseradish peroxidase-labeled secondary antibody, and a dye. The primary antibody includes an antiphosphatidylinositol proteoglycan 3 antibody; The secondary antibody specifically recognizes the primary antibody.

[0032] This kit diagnoses pleural pulmonary blastoma by detecting the expression level of phosphatidylinositol proteoglycan 3 in the test sample.

[0033] In some alternative embodiments, the dye includes, but is not limited to, hematoxylin.

[0034] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0035] Example 1 1.1 Expression and distribution of GPC3 in tumor tissues and adjacent tissues: All surgical pathological tissues (formalin fixed, paraffin embedded) were obtained from Guangzhou Women and Children's Medical Center, including 25 cases of PPB (disease group), 5 cases of adjacent normal tissue (normal control group), and 10 cases of congenital cystic lung lesions (disease control group). All tissues were fixed in 10% formaldehyde, embedded in paraffin, and cut into 4 μm thick sections. Hematoxylin and eosin (H&E) and immunohistochemical staining were then performed. For H&E staining, sections were stained with hematoxylin for 5 minutes, followed by eosin staining for 3 minutes. For immunohistochemical staining, sections were incubated overnight at 4°C with GPC3 monoclonal antibody (ab207080, Abcam). Subsequently, sections were incubated with HRP-labeled goat anti-rabbit IgG (ab6721, Abcam) at 4°C for 20 minutes. Cell nuclei were stained with hematoxylin staining solution. The stained sections were examined under an optical microscope at 10x and 40x magnification, and the results are as follows: Figure 1 (In the image, PPB represents pleural pulmonary blastoma; Adjacent tissue represents adjacent normal tissue; CPAM represents congenital cystic pulmonary lesions; the first and second columns on the left are stained sections examined under an optical microscope at 10x and 40x magnification, respectively.) It can be seen that GPC3 expression is significantly upregulated in the pathological tissue of pleural pulmonary blastoma.

[0036] Subsequently, the intensity of GPC3 immunomarking was scored. Based on the immunohistochemical expression level of GPC3 in PPB, the cases were divided into three groups. Immunohistochemical staining showed that GPC3 expression was present in all 25 PPB patients, with varying immunomarking densities, categorized as weak (+, 11 / 25), moderate (++, 4 / 25), and strong (+++, 10 / 25), as shown in Table 1.

[0037] Table 1. Expression of GPC3 in the pleural pulmonary blastoma disease cohort.

[0038] Note: M = male, F = female. L = left, R = right. "Y" means the event has already occurred. "N" means the event has not yet occurred. "-" means there has been no death.

[0039] 1.2 Enzyme-linked adsorption spectroscopy (ELISA) was used to detect the expression level of GPC3 serum protein: Preoperative venous blood (1 mL) was collected from 10 patients with PPB disease (disease group), 5 patients with congenital cystic lung disease (disease control group), and 5 age- and sex-matched children with non-pulmonary diseases (normal group). The blood was placed in anticoagulant tubes containing EDTA, allowed to stand at 4°C for 2 hours, centrifuged at 1000g for 15 minutes, and the supernatant was collected, labeled, and stored at -80°C. The kit used a quantitative double-antibody sandwich enzyme-linked immunosorbent assay (Invitrogen). Sensitivity: The minimum detectable dose of human GPC3 was 0.8 ng / mL. Specificity: No significant cross-reaction or interference was observed between human GPC3 and analogues. Precision between different wells of the same dropper plate: Coefficient of variation (CV%) < 8%. The serum GPC3 protein expression levels of the disease group, disease control group, and normal group were detected by enzyme-linked adsorption assay (ELISA). The results are shown in Table 2 (PPB, pleural pulmonary blastoma; Cont, age- and sex-matched non-pulmonary disease children; CPAM, congenital cystic pulmonary lesion).

[0040] Table 2

[0041] The results showed that serum GPC3 was significantly elevated in the PPB group (n=10), while it was low in the control group (Cont) and the CPAM group. ROC curve analysis showed that GPC3 had high diagnostic value for PPB (AUC=1, P<0.001, Table 2). When the serum GPC3 concentration was >30 ng / mL, the sensitivity for diagnosing PPB was 90% (95% CI: 72.25%-100%), and the specificity was 100% (95% CI: 59.58%-99.49%).

[0042] Example 2 2.1 Relationship between differential GPC3 expression in pleural pulmonary blastoma and survival prognosis: A retrospective analysis compared data from 25 patients with polycystic purpura (PPB) who underwent surgery at Guangzhou Women and Children's Medical Center between 2014 and 2022 (the disease group in Example 1), with a 2-year follow-up. Staining intensity was assessed as: [-, negative; +, low intensity; ++, moderate intensity; +++, high intensity]. The immunohistochemical expression level of GPC3 in PPB was divided into three groups: low expression (GPC3+), moderate expression (GPC3++), and high expression (GPC3+++). Survival curves for each group were then analyzed based on GPC3 expression levels. Survival analysis was performed using Kaplan-Meier (Log-rank) statistics. Figure 2 ).

[0043] Figure 2Figure A shows the assessment of GPC3 expression levels in 25 PPB cases using immunohistochemical staining, and the scoring based on the intensity of GPC3 immunomarking. Figure 2 B in the data shows that all 25 PPB cases were divided into three groups based on GPC3 expression. Figure 2 The C value in the figure represents the overall survival of the three groups after 24 months of follow-up. The p-value after the log-rank test was 0.0035, indicating a difference in survival rates among the three curves. Red represents the GPC3+++ group, green represents the GPC3++ group, and blue represents the GPC3+ group.

[0044] Based on the above analysis, it is suggested that GPC3 can serve as a highly specific indicator for the pathological diagnosis of PPB disease.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of reagents for detecting phosphatidylinositol proteoglycan 3 in the preparation of products for diagnosing pleural pulmonary blastoma.

2. The application according to claim 1, characterized in that, The reagents are used to test samples including lung tissue and blood.

3. The application according to claim 1, characterized in that, The expression of phosphatidylinositol proteoglycan 3 was upregulated in pleural pulmonary blastoma.

4. The application according to claim 1, characterized in that, The product includes a reagent kit.

5. Application of reagents for detecting phosphatidylinositol proteoglycan 3 in the preparation of products for assessing the prognosis of pleural pulmonary blastoma.

6. The application according to claim 5, characterized in that, The reagents are used to test samples including lung tissue and blood.

7. The application according to claim 5, characterized in that, The expression of phosphatidylinositol proteoglycan 3 was upregulated in pleural pulmonary blastoma.

8. The application according to claim 5, characterized in that, The product includes a reagent kit.

9. A kit for diagnosing or assessing the prognosis of pleural pulmonary blastoma, characterized in that, The kit is based on immunohistochemical staining to detect the expression level of phosphatidylinositol proteoglycan 3, and includes a primary antibody, a horseradish peroxidase-labeled secondary antibody, and a dye. The primary antibody includes an antiphosphatidylinositol proteoglycan 3 antibody; The secondary antibody specifically recognizes the primary antibody.

10. The reagent kit according to claim 9, characterized in that, The dyes include hematoxylin.

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