Application of reagent for detecting AP3S2 in preparation of reagent for diagnosing neoadjuvant immunochemotherapy resistance of gastric cancer in local development stage

Abstract

The invention discloses an application of a reagent for detecting AP3S2 in preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance of gastric cancer in a local progression stage, which comprises the following steps: screening differential expression proteins in neoadjuvant immunochemotherapy resistance tissues of gastric cancer in the local progression stage through proteomics; a new marker is provided for diagnosis of the local progression stage gastric cancer immunochemotherapy drug resistance, and a basis is also provided for development and future development of a local progression stage gastric cancer immunochemotherapy drug resistance diagnosis method.

Description

Technical Field

[0001] This invention relates to the field of detection, and more specifically to the application of reagents for detecting AP3S2 in the preparation of diagnostic reagents for neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer. Background Technology

[0002] Gastric cancer (GC) is a highly aggressive malignant tumor, with approximately 480,000 new cases and 370,000 deaths annually in my country. GC accounts for 44% of the global burden and is a major malignant tumor threatening the health of Chinese residents. The 5-year survival rate for early-stage GC (T1N0M0) is approximately 95%, while the median survival for patients with advanced / metastatic GC is only 9-10 months. 70% of patients are diagnosed at a locally advanced (LAGC) or metastatic stage. LAGC is defined as GC / gastroesophageal junction adenocarcinoma with clinical stage T3-4 (tumor infiltrating the serosa or adjacent structures) and / or regional lymph node metastasis (N+), but no distant metastasis (M0), accounting for approximately 50-60% of operable patients. Immune checkpoint blockade (ICB) has opened a new era of hope for GC immunotherapy. Compared to chemotherapy, regardless of PD-L1 positivity score greater than 5, the immune checkpoint inhibitor nivolumab combined with chemotherapy as first-line treatment improves overall survival (OS) and progression-free survival (PFS) in patients with locally advanced gastric adenocarcinoma or gastroesophageal junction adenocarcinoma. Due to the progress made in immunotherapy for advanced gastric / gastroesophageal cancer (GC / GEJ), an increasing number of studies are exploring neoadjuvant therapy. However, not all patients with locally advanced gastric cancer benefit from neoadjuvant ICB, and some may even experience increased risk of disease progression, treatment-related adverse reactions, increased surgical difficulty, or increased risk of postoperative complications. Therefore, it is necessary to explore potential mechanisms and related biomarkers to screen GC patients who may benefit from immunotherapy.

[0003] Establishing effective diagnostic methods for resistance to immunotherapy combined with chemotherapy in locally advanced gastric cancer is crucial for improving the diagnosis and treatment of locally advanced gastric cancer, reducing recurrence and metastasis, and improving long-term survival. However, effective biomarkers are currently lacking.

[0004] Discovering and identifying biomarkers is the foundation for establishing a diagnostic method. Therefore, the identification and application of new biomarkers is the fundamental work for promoting the development of disease diagnosis and treatment methods.

[0005] This invention, through high-precision spatial proteomics research and subsequent studies, discovered that the adapter-related protein complex 3 subunit σ2 (AP3S2) possesses the property of serving as a biomarker for diagnosing immunochemotherapy resistance in locally advanced gastric cancer. Therefore, AP3S2 detection reagents can be used to prepare diagnostic reagents for drug resistance in locally advanced gastric cancer. This invention provides a new strategy for the development of diagnostic methods for drug resistance in locally advanced gastric cancer. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide an application of a reagent for detecting AP3S2 in the preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer.

[0007] To achieve the above objectives, the present invention provides the following technical solution: 1. Application of AP3S2 detection reagent in the preparation of diagnostic reagent for neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer, wherein the neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer refers to the resistance of primary locally advanced gastric cancer cells to immune checkpoint inhibitors combined with chemotherapy drugs.

[0008] Preferably, the locally advanced gastric cancer of this invention refers to GC / gastroesophageal junction adenocarcinoma with clinical stage T3-4 and / or regional lymph node metastasis, but without distant metastasis.

[0009] Preferably, the ID of the AP3S2 in the NCBI database is 10239, and its ID in the Uniprot database is P59780.

[0010] Preferably, the reagent used to detect AP3S2 is for detecting the expression level of the AP3S2 protein.

[0011] Preferably, the reagents for detecting AP3S2 in this invention employ enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay, immunoprecipitation assay, immunoblotting, high-performance liquid chromatography (HPLC), capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence assay, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance (SPR), immunoPCR, or biotinylate assay.

[0012] Preferably, the reagent used to detect AP3S2 in this invention is an immunohistochemical detection method.

[0013] The beneficial effects of this invention are as follows: This invention provides the application of reagents for detecting AP3S2 in the preparation of diagnostic reagents for neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer. By screening differentially expressed proteins in epithelial tissues of locally advanced gastric cancer resistant to neoadjuvant immunochemotherapy through spatial proteomics, it provides new biomarkers for the diagnosis of immunochemotherapy resistance in locally advanced gastric cancer, and also lays the foundation for the development and future advancement of diagnostic methods for immunochemotherapy resistance in locally advanced gastric cancer. Attached Figure Description

[0014] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 To classify locally advanced gastric cancer cases into chemotherapy-combined immune checkpoint inhibitor-resistant and sensitive groups based on tumor regression grade, spatial proteomics was used to divide the fibrous-cut epithelial / mesenchymal regions. Figure 2 Differences in protein profiles of surgically resected tissues from locally advanced gastric cancer cases that were sensitive to and resistant to immunochemotherapy. Figure 3 The relative mass spectrometry values ​​of AP3S2 in surgically resected tissues from locally advanced gastric cancer cases sensitive and resistant to neoadjuvant immunochemotherapy. Figure 4 Immunohistochemical staining images of representative samples of surgically resected tissue from locally advanced gastric cancer cases that were sensitive and resistant to immunochemotherapy; Figure 5 The immunohistochemical score of AP3S2 in the training set (n=241) of surgically resected tissues from locally advanced gastric cancer cases that were sensitive and resistant to immunochemotherapy. Figure 6 The immunohistochemical score of AP3S2 in the validation set of surgically resected tissues (n=81) of locally advanced gastric cancer cases that were sensitive and resistant to immunochemotherapy. Figure 7 To investigate the accuracy of AP3S2 as a biomarker in differentiating the sensitivity of locally advanced gastric cancer to immunochemotherapy in the training set (n=241); Figure 8 To investigate the accuracy of AP3S2 as a biomarker in differentiating the sensitivity of locally advanced gastric cancer to immunochemotherapy in a validation set (n=81). Detailed Implementation

[0015] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0016] Example 1: Screening for biomarkers of immunochemotherapy resistance in locally advanced gastric cancer using proteomics analysis strategies. The main drugs used in immunochemotherapy for locally advanced gastric cancer include chemotherapeutic agents such as cisplatin combined with paclitaxel, and immune checkpoint inhibitors such as sintilimab (an anti-PD-1 monoclonal antibody). Residual tissue after neoadjuvant immunochemotherapy tends to accumulate drug-resistant cancer cells. This study utilizes this characteristic to screen for biomarkers of immunochemotherapy resistance by comparing the protein profiles of surgically resected tissues from immunochemosensitive and drug-resistant cases of locally advanced gastric cancer. Specifically, three immunochemosensitive and three drug-resistant tissue samples were collected. High-precision spatial proteomics technology was used to detect protein expression levels at the proteomic level in both tissues, and differentially expressed proteins in the drug-resistant samples were then compared and screened. The spatial proteomic regional fibrosis cleavage scheme is as follows: Figure 1 As shown, the differences in protein maps between the two tissues are as follows: Figure 2 As shown, the detection signal value of AP3S2 was significantly increased in the drug-resistant samples ( Figure 3 ).

[0017] The method for obtaining samples of locally advanced gastric cancer tissue after immunochemotherapy and the method for detecting the high-precision spatial proteomics in this embodiment are as follows: [1] Haitao Huang, Na Li, Yingkuan Liang, Rutao Li, XingTong, Jinyuan Xiao, Hongzhen Tang, Dong Jiang, Kai Xie, Chen Fang, ShaomuChen, Guangbin Li, Bin Wang, Jiaqian Wang, Haitao Luo, Lingchuan Guo, HaitaoMa, Wei Jiang, Yu Feng. Multi-omics analyses reveal spatial heterogeneity in primary and metastatic oesophageal squamous cell carcinoma. Clin Transl Med. 2023 Nov;13(11): e1493. doi:10.1002 / ctm2.1493. PMID: 38009315 PMCID:PMC10679972.and [2] Thierry M Nordmann, Holly Anderton, Akito Hasegawa, LisaSchweizer, Peng Zhang, Pia-Charlotte Stadler, Ankit Sinha, Andreas Metousis, Florian A Rosenberger, Maximilian Zwiebel, Takashi K Satoh, FlorianAnzengruber, Maximilian T Strauss, Maria C Tanzer, Yuki Saito, Ting Gong, Marvin Thielert, Haruna Kimura, Natasha Silke, Edwin H Rodriguez, Gaetana Restivo, Hong Ha Nguyen, Annette Gross, Laurence Feldmeyer, Lukas Joerg, Mitchell P Levesque, Peter J Murray, Saskia Ingen-Housz-Oro, Andreas Mund, Riichiro Abe, John Silke, Chao Ji, Lars E French, Matthias Mann. Spatialproteomics identifies JAKi as treatment for a lethal skin disease. Nature.2024 Nov; 635(8040):1001-1009. doi: 10.1038 / s41586-024-08061-0. Epub 2024 Oct16. PMID: 39415009 PMCID: PMC11602713. Example 2: Verification of increased AP3S2 expression in locally advanced gastric cancer tissues resistant to immunochemotherapy In the aforementioned discovery phase, the measured values ​​of each protein only represent their average values ​​across three samples, which may be subject to chance. Therefore, to verify the nature of the increased expression of AP3S2 in immunochemotherapy-resistant tissues discovered in the discovery phase, this study further examined 146 resistant samples (treatment response rate scores TGR3, 4, and 5) and 95 sensitive samples (treatment response rate scores TGR1 and 2) using clinically common immunohistochemistry techniques. The results are as follows: Figures 4-6As shown in the figure. The results indicate that this experiment further demonstrated the high expression of AP3S2 in immunochemotherapy-resistant tissues of locally advanced gastric cancer in both the training and validation sets. Therefore, AP3S2 possesses the fundamental properties to serve as a biomarker for immunochemotherapy resistance in locally advanced gastric cancer.

[0018] The immunohistochemical procedures and results interpretation methods in this embodiment are described in Md Khurshidul Hassan, Dinesh Kumar, Saket Awadhesbhai Patel, Niharika Pattanaik, NachiketaMohapatra, Manjusha Dixit. Expression pattern of EEF1A2 in brain tumors: Histological analysis and functional role as a promoter of EMT. Life Sci. 2020 Apr 1:246:117399. doi: 10.1016 / j.lfs.2020.117399. Epub 2020 Feb 4. PMID:32032648 DOI: 10.1016 / j.lfs.2020.117399.

[0019] Example 3: AUC value of AP3S2 protein expression level in differentiating neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer AUC (Acceptance Value of Criteria) is a scientifically recognized method in the diagnostic field for measuring the accuracy of biomarkers. To measure the accuracy of AP3S2 in diagnosing neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer, this study plotted ROC curves using data from Example 2. The AUC values ​​in the training and validation sets were 0.8642 and 0.8700, respectively. Figure 7 (8). Based on the data, it can be concluded that when the threshold is set at 6.3 for AP3S2 protein expression in tissues detected by immunohistochemistry, patients with locally advanced gastric cancer resistant to neoadjuvant immunochemotherapy can be diagnosed with this threshold. The sensitivity of this diagnosis is 82.11%, and the specificity is 79.45%. In conclusion, AP3S2 can be used as a biomarker for diagnosing locally advanced gastric cancer resistant to immunochemotherapy, and AP3S2 detection reagents have applications in preparing diagnostic reagents for drug resistance in locally advanced gastric cancer.

[0020] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The application of a reagent for detecting AP3S2 in the preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer, characterized in that: The neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer refers to the resistance of primary locally advanced gastric cancer cells to immune checkpoint inhibitors combined with chemotherapy drugs.

2. The application of the reagent for detecting AP3S2 according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer, characterized in that: The term "locally advanced gastric cancer" refers to gastric cancer with clinical stage T3-4 and / or regional lymph node metastasis, but without distant metastasis, specifically gastric / gastroesophageal junction adenocarcinoma.

3. The application of the reagent for detecting AP3S2 according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer, characterized in that: The AP3S2 has the ID 10239 in the NCBI database and the ID P59780 in the Uniprot database.

4. The application of the reagent for detecting AP3S2 according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer, characterized in that: The reagent used to detect AP3S2 is for detecting the expression level of the AP3S2 protein.

5. The application of the reagent for detecting AP3S2 according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer, characterized in that: The reagents used to detect AP3S2 include enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay, immunoprecipitation assay, immunoblotting, high performance liquid chromatography (HPLC), capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence assay, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance (SPR), immunoPCR, or biotinylate assay.

6. The application of the reagent for detecting AP3S2 according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer, characterized in that: The reagents used to detect AP3S2 were obtained using an immunohistochemical detection method.

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