Application of reagent for detecting PLS1 in preparation of reagent for diagnosing neoadjuvant immunochemotherapy resistance of gastric cancer in local development stage

Abstract

The invention discloses application of a reagent for detecting PLS1 in preparation of a reagent for diagnosing neoadjuvant immunochemotherapy resistance of gastric cancer in a local progression stage, differentially expressed proteins in neoadjuvant immunochemotherapy resistance tissues of gastric cancer in the local progression stage are screened out through proteomics, and a new marker is provided for diagnosis of neoadjuvant immunochemotherapy resistance of gastric cancer in the local progression stage. And a foundation is provided for the development and future development of a diagnosis method for the immunochemotherapy drug resistance of the gastric cancer in the local development stage.

Description

TECHNICAL FIELD

[0001] The application relates to the detection field and particularly relates to application of a reagent for detecting PLS1 in preparation of a locally advanced gastric cancer neoadjuvant immunotherapy drug resistance diagnostic reagent. BACKGROUND

[0002] Gastric cancer (GC) is a highly invasive malignant tumor. There are about 480,000 new cases and 370,000 deaths in China every year. The global GC burden of 44% is a major malignant tumor threatening the health of Chinese residents. The 5-year survival rate of early GC (T1N0M0) is about 95%, while the median survival of patients with advanced / metastatic GC is only 9-10 months. 70% of patients are diagnosed as locally advanced (LAGC) or metastatic stage. LAGC is defined as GC / gastroesophageal junction adenocarcinoma with clinical stage T3-4 (tumor infiltration serosa or adjacent structure) and / or regional lymph node metastasis (N+) but no distant metastasis (M0), accounting for about 50-60% of patients who can be operated. Immune checkpoint blockade (ICB) has opened a new era of GC immunotherapy. Compared with chemotherapy, the immune checkpoint inhibitor nivolumab combined with chemotherapy as first-line treatment can improve the overall survival (OS) and progression-free survival (PFS) of patients with locally advanced gastric adenocarcinoma or gastroesophageal junction adenocarcinoma, regardless of whether the PD-L1 positive score is greater than 5. Due to the progress of immunotherapy in advanced gastric cancer / gastroesophageal cancer (GC / GEJ), more and more studies have begun to explore neoadjuvant therapy. However, not all patients with locally advanced gastric cancer can benefit from neoadjuvant ICB, and even increase the risk of disease progression, treatment-related adverse reactions, increase the difficulty of surgery or increase the risk of postoperative complications. Therefore, it is necessary to explore the potential mechanism and related markers to screen GC patients who may benefit from immunotherapy.

[0003] Establishing an effective diagnosis method for immunotherapy combined with chemotherapy resistance of locally advanced gastric cancer is crucial to improve the diagnosis and treatment level of locally advanced gastric cancer, reduce recurrence and metastasis, and improve long-term survival rate. However, there is still a lack of effective markers.

[0004] Finding and identifying markers is the basis for establishing a diagnostic method. Therefore, the identification and application of new markers are the fundamental work to promote the development of disease diagnosis and treatment methods.

[0005] The application discloses a method for diagnosing drug resistance of locally advanced gastric cancer, and belongs to the field of tumor drug resistance diagnosis. SUMMARY

[0006] Therefore, one of the purposes of the application is to provide an application of a PLS1 detection reagent in preparation of a locally advanced gastric cancer neoadjuvant immunochemo-therapy drug resistance diagnostic reagent.

[0007] To achieve the above-mentioned purposes, the application provides the following technical solutions. The application of the PLS1 detection reagent in preparation of a locally advanced gastric cancer neoadjuvant immunochemo-therapy drug resistance diagnostic reagent, wherein the locally advanced gastric cancer neoadjuvant immunochemo-therapy drug resistance refers to drug resistance of primary locally advanced gastric cancer cells to an immune checkpoint inhibitor combined with a chemotherapeutic drug.

[0008] Preferably, the locally advanced gastric cancer refers to a GC / gastroesophageal junction adenocarcinoma disease in a clinical stage T3-4 (tumor infiltration serosa or adjacent structure) and / or regional lymph node metastasis (N+) but without distant metastasis (M0).

[0009] Preferably, the PLS1 has an ID of 5357 in a NCBI database and an ID of Q14651 in a Uniprot database.

[0010] Preferably, the PLS1 detection reagent is used for detecting a PLS1 protein expression level.

[0011] Preferably, the PLS1 detection reagent is an enzyme-linked immunosorbent assay, an immunofluorescence method, a radioimmunoassay method, an immunoprecipitation method, an immunoblotting method, a high performance liquid chromatography method, a capillary gel electrophoresis method, a near-infrared spectroscopy method, a mass spectrometry method, an immunochemiluminescence method, a colloidal gold immunological technology, a fluorescent immunochromatography technology, a surface plasmon resonance technology, an immunological PCR technology or a biotin avidin technology.

[0012] Preferably, the PLS1 detection reagent is an immunohistochemical detection method.

[0013] The application has the beneficial effects that the application provides application of a reagent for detecting PLS1 in preparation of a new adjuvant immunotherapy and chemotherapy drug resistance diagnostic reagent for locally advanced gastric cancer, and differentially expressed proteins in epithelial tissue of the new adjuvant immunotherapy and chemotherapy drug resistance of locally advanced gastric cancer are screened through spatial proteomics, a new marker is provided for immunotherapy and chemotherapy drug resistance diagnosis of locally advanced gastric cancer, and a foundation is provided for development and future development of the immunotherapy and chemotherapy drug resistance diagnosis method of locally advanced gastric cancer. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to make the objectives, technical solutions and beneficial effects of the application clearer, the application provides the following drawings for description: Figure 1 The fibro-cut epithelial / interstitial region of the spatial proteomics is divided based on tumor regression grade to divide the locally advanced gastric cancer cases into a chemotherapy combined with an immune checkpoint inhibitor treatment drug resistance group and a sensitive group; Figure 2 The protein atlas difference of the surgical resection tissue of the locally advanced gastric cancer immunotherapy and chemotherapy sensitive and drug resistant cases; Figure 3 The mass spectrometry detection relative value of PLS1 in the surgical resection tissue of the locally advanced gastric cancer new adjuvant immunotherapy and chemotherapy sensitive and drug resistant cases; Figure 4 The representative sample immunohistochemical staining image of PLS1 in the surgical resection tissue of the locally advanced gastric cancer immunotherapy and chemotherapy sensitive and drug resistant cases; Figure 5 The immunohistochemical score of PLS1 in the training set (n=241) of the surgical resection tissue of the locally advanced gastric cancer immunotherapy and chemotherapy sensitive and drug resistant cases; Figure 6 The immunohistochemical score of PLS1 in the verification set (n=81) of the surgical resection tissue of the locally advanced gastric cancer immunotherapy and chemotherapy sensitive and drug resistant cases; Figure 7 The accuracy research of PLS1 as a marker in the training set (n=241) for distinguishing the immunotherapy and chemotherapy sensitivity of locally advanced gastric cancer; Figure 8 The accuracy research of PLS1 as a marker in the verification set (n=81) for distinguishing the immunotherapy and chemotherapy sensitivity of locally advanced gastric cancer. DETAILED DESCRIPTION

[0015] The application will be further described below in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the application and implement it, but the embodiments are not limiting to the application.

[0016] Example 1: Screening of an immunotherapy and chemotherapy drug resistance marker for locally advanced gastric cancer by proteomics analysis strategy The main drugs for immunotherapy of locally advanced gastric cancer include chemotherapy drugs represented by cisplatin combined with paclitaxel, and immune checkpoint inhibitors represented by sintilimab. The residual tissue after neoadjuvant immunotherapy will form an enrichment of drug-resistant cancer cells. This study takes advantage of this feature by comparing the protein profiles of locally advanced gastric cancer immunotherapy sensitive and resistant cases to screen markers for immunotherapy resistance. The specific content is as follows: 3 cases of immunotherapy sensitive and resistant tissues were collected, the protein expression level in the proteome of the two kinds of tissues was detected by high-precision spatial proteomics technology, and the differentially expressed proteins in the resistant samples were screened and compared. The spatial proteomic region fiber cutting scheme is shown in Figure 1 , and the protein profile difference of the two kinds of tissues is shown in Figure 2 , wherein the detection signal value of PLS1 in the resistant sample is significantly increased ( Figure 3 ).

[0017] The methods of sample collection and high-precision spatial proteomics technology detection of locally advanced gastric cancer tissue samples after immunotherapy in this example are described in: [1] Haitao Huang, Na Li, Yingkuan Liang, Rutao Li, Xing Tong, Jinyuan Xiao, Hongzhen Tang, Dong Jiang, Kai Xie, Chen Fang, Shaomu Chen, Guangbin Li, Bin Wang, Jiaqian Wang, Haitao Luo, Lingchuan Guo, Haitao Ma, 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, Lisa Schweizer, Peng Zhang, Pia-Charlotte Stadler, Ankit Sinha, Andreas Metousis, Florian A Rosenberger, Maximilian Zwiebel, Takashi K Satoh, Florian Anzengruber, 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. Spatial proteomics 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 Oct 16. PMID: 39415009 PMCID: PMC11602713. Example 2, Verification of the elevated expression of PLS1 in immunochemotherapy-resistant tissues of locally advanced gastric cancer In the foregoing discovery phase, the detection value of each protein only represents its average value in 3 samples, and there may be randomness. Therefore, in order to verify the nature of the elevated expression of PLS1 in immunochemotherapy-resistant tissues discovered in the discovery phase, the present study further detected 146 cases of drug resistance (treatment response rate score TGR3, 4, 5) and 95 cases of sensitivity (treatment response rate score TGR1, 2), and the detection method was the commonly used immunohistochemical technique in clinical practice, and the results were as follows Figures 4-6The results show that the experiment further proves the high expression of PLS1 in the immunotherapy and chemotherapy-resistant tissues of locally advanced gastric cancer in the training set and the validation set, respectively. Therefore, PLS1 has the basic properties of a marker for immunotherapy and chemotherapy resistance of locally advanced gastric cancer.

[0018] The immunohistochemical operation and result interpretation method in this embodiment are described in Md Khurshidul Hassan, Dinesh Kumar, Saket Awadhesbhai Patel, Niharika Pattanaik, Nachiketa Mohapatra, 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 PLS1 protein expression level for identifying neoadjuvant immunotherapy and chemotherapy resistance of locally advanced gastric cancer The AUC value is a scientific method recognized in the diagnosis field to measure the accuracy of a marker. To measure the accuracy of PLS1 in diagnosing neoadjuvant immunotherapy and chemotherapy resistance of locally advanced gastric cancer, the ROC curve was drawn by the data of Example 2, and the AUC values in the training set and the validation set were 0.9062 and 0.8990, respectively. Figure 7 According to the data, when the threshold value is set to 6.9 for the protein expression level of PLS1 in the tissue detected by immunohistochemistry, it can be judged that the patient is a neoadjuvant immunotherapy and chemotherapy-resistant patient with locally advanced gastric cancer if the threshold value is higher than the threshold value. The sensitivity of this judgment is 86.32%, and the specificity is 78.77%. In summary, PLS1 can be used as a marker for diagnosing immunotherapy and chemotherapy-resistant locally advanced gastric cancer, and the PLS1 detection reagent has the application of preparing a locally advanced gastric cancer drug resistance diagnostic reagent.

[0020] The above-described embodiments are only preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art based on the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. The application of reagents for detecting PLS1 in the preparation of diagnostic reagents 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 PLS1 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 GC / gastroesophageal junction adenocarcinoma with clinical stage T3-4 (tumor infiltrating the serosa or adjacent structures) and / or regional lymph node metastasis (N+), but without distant metastasis (M0).

3. The application of the reagent for detecting PLS1 according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance in locally advanced gastric cancer, characterized in that: The PLS1 has an ID of 5357 in the NCBI database and an ID of Q14651 in the Uniprot database.

4. The application of the reagent for detecting PLS1 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 PLS1 is for detecting the expression level of the PLS1 protein.

5. The application of the reagent for detecting PLS1 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 PLS1 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 PLS1 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 PLS1 were obtained using an immunohistochemical detection method.

Images