Application of reagent for detecting PEDS1 in preparation of neoadjuvant immunochemotherapy drug resistance diagnostic reagent for local advanced esophageal squamous carcinoma

Abstract

The invention discloses application of a reagent for detecting PEDS1 in preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance of local advanced esophageal squamous carcinoma. Differential expression proteins in neoadjuvant immunochemotherapy resistance tissues of local advanced esophageal squamous carcinoma are screened out through proteomics; a new marker is provided for diagnosis of local advanced esophageal squamous cell carcinoma immunochemotherapy drug resistance, and a basis is also provided for development and future development of a local advanced esophageal squamous cell carcinoma immunochemotherapy drug resistance diagnosis method.

Description

Technical Field

[0001] The present invention relates to the field of detection, and in particular to the use of a reagent for detecting PEDS1 in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in locally advanced esophageal squamous cell carcinoma. Background Art

[0002] Esophageal cancer (EC) is a common malignancy worldwide and a major health threat to Chinese residents. In China, over 90% of esophageal cancers are esophageal squamous cell carcinoma (ESCC). Surgery alone has a low overall survival (OS) rate and a high five-year recurrence rate. Although neoadjuvant chemoradiotherapy (NCRT) or neoadjuvant chemotherapy (NCT) combined with surgical resection can improve survival in patients with locally advanced EC, the prognosis remains poor for some patients. In recent years, PD-1 inhibitors have become a popular cancer immunotherapy by blocking the interaction between PD-1 and PD-L1, restoring the anti-tumor activity of T lymphocytes, enhancing immune responses, and reducing tumor cell proliferation and metastasis. Immunochemotherapy (ICT) has become the standard first-line treatment for advanced EC, providing significant survival benefits for patients. Whether neoadjuvant immunochemotherapy (NICT) can offer patients with locally advanced EC greater opportunities for cure has become a key research topic. Currently, multiple clinical studies targeting neoadjuvant immunotherapy for EC are underway, with impressive results. Sintilimab is a fully humanized, recombinant IgG4 monoclonal antibody targeting PD-1. It offers practical advantages, including relatively low treatment costs and is considered safe and effective in clinical settings. Although multiple studies have confirmed the clinical efficacy of neoadjuvant sintilimab in the treatment of EC, some patients fail to benefit from NICT and may even experience an increased risk of disease progression, treatment-related adverse reactions, increased surgical difficulty, or an increased risk of postoperative complications.

[0003] Establishing an effective diagnostic method for NICT resistance in locally advanced esophageal squamous cell carcinoma is crucial to improving the diagnosis and treatment of esophageal squamous cell carcinoma, reducing recurrence and metastasis, and improving long-term survival rate, but there is currently a lack of effective markers. Summary of the Invention

[0004] In light of this, one of the objectives of the present invention is to provide a reagent for detecting PEDS1 for use in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in locally advanced esophageal squamous cell carcinoma. Through high-precision spatial proteomics research and subsequent studies, the present invention discovered that plasma acetylethanolamine desaturase 1 (PEDS1) possesses the properties of a diagnostic marker for immunotherapy resistance in locally advanced esophageal squamous cell carcinoma. Therefore, a PEDS1 detection reagent can be used in the preparation of a diagnostic reagent for drug resistance in locally advanced esophageal squamous cell carcinoma. This invention provides a new strategy for the development of methods for diagnosing and treating drug resistance in locally advanced esophageal squamous cell carcinoma.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: The use of a reagent for detecting PEDS1 in the preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma, wherein the patient's resistance to a neoadjuvant immunochemotherapy regimen comprising immune checkpoint inhibitors and chemotherapy drugs is determined by detecting the expression level of PEDS1 protein in esophageal squamous cell carcinoma tissue, wherein the locally advanced esophageal squamous cell carcinoma is staged as cT3-4aN0 / +M0 or cT1b-2N+M0.

[0006] Preferably, the detection object of the present invention is a tissue sample of esophageal squamous cell carcinoma surgically resected after neoadjuvant immunochemotherapy.

[0007] Preferably, the PEDS1 has an ID of 387521 in the NCBI database and an ID of A5PLL7 in the Uniprot database.

[0008] Preferably, the reagent for detecting PEDS1 adopts enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, immunocoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunoassay, fluorescence immunochromatography, surface plasmon resonance, immuno-PCR or biotin-avidin technology.

[0009] Preferably, the detection is performed using immunohistochemistry.

[0010] Preferably, in the present invention, when the QRSL1 protein expression level in immunohistochemistry is ≥5.7, the patient is determined to have drug resistance.

[0011] The beneficial effects of the present invention are as follows: the present invention provides an application of a reagent for detecting PEDS1 in the preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma, and screens out differentially expressed proteins in neoadjuvant immunochemotherapy-resistant tissues of locally advanced esophageal squamous cell carcinoma through proteomics, providing a new marker for the diagnosis of immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma, and also providing a basis for the development and future development of a diagnostic method for immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration: Figure 1 To conduct spatial proteomics analysis to stratify esophageal squamous cell carcinoma cases into chemotherapy combined with immune checkpoint inhibitor treatment-resistant and sensitive groups based on tumor regression grade; Figure 2 To investigate the differences in protein profiles of surgically resected tissues in patients with locally advanced esophageal squamous cell carcinoma who are sensitive to immunochemotherapy and those who are resistant; Figure 3 The relative value of PEDS1 detected by mass spectrometry in surgical resection tissues of locally advanced esophageal squamous cell carcinoma with neoadjuvant immunotherapy sensitivity and resistance; Figure 4 Representative immunohistochemical staining images of PEDS1 in surgically resected tissues of locally advanced esophageal squamous cell carcinoma with chemoimmunosensitive and resistant conditions. Figure 5 To evaluate the immunohistochemical expression of PEDS1 in a training set of surgically resected tissues from patients with locally advanced esophageal squamous cell carcinoma (ESCC) who were sensitive to immunochemotherapy and those who were resistant to it (n=188). Figure 6 To evaluate the immunohistochemical score of PEDS1 in a validation set of surgical resection tissues from locally advanced esophageal squamous cell carcinoma (ESCC) with chemoimmunotherapy sensitivity and resistance (n=83). Figure 7 To investigate the accuracy of PEDS1 as a biomarker for distinguishing the sensitivity of locally advanced esophageal squamous cell carcinoma to immunochemotherapy in a training set (n=188); Figure 8 To investigate the accuracy of PEDS1 as a marker for distinguishing the sensitivity of locally advanced esophageal squamous cell carcinoma to immunochemotherapy in a validation set (n=83). DETAILED DESCRIPTION

[0013] 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 the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0014] Example 1: Screening for immunochemotherapy resistance markers in locally advanced esophageal squamous cell carcinoma using a proteomic analysis strategy The main drugs for immunochemotherapy of locally advanced esophageal squamous cell carcinoma include chemotherapy drugs represented by cisplatin combined with paclitaxel, and immune checkpoint inhibitors represented by Sindily anti-PD1 monoclonal antibody. The residual tissue after neoadjuvant immunochemotherapy will form an enrichment of drug-resistant cancer cells. This study took advantage of this feature and screened markers of immunochemotherapy resistance by comparing the protein profiles of surgically resected tissues of locally advanced esophageal squamous cell carcinoma that were sensitive to immunochemotherapy and those that were resistant. The specific contents are as follows: 3 cases of immunochemotherapy-sensitive and resistant tissues were collected, and high-precision spatial proteomics technology was used to detect the protein expression levels at the proteomic level in the two tissues, and then the differentially expressed proteins in the resistant samples were compared and screened. The spatial proteomic regional fiber cutting scheme is as follows: Figure 1 The protein profiles of the two tissues differ as shown in Figure 2 As shown, the detection signal value of PEDS1 in drug-resistant samples was significantly increased ( Figure 3 ).

[0015] For the methods of sampling esophageal squamous cell carcinoma tissue samples after immunochemotherapy and high-precision spatial proteomics detection in this example, please refer to: [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, 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 elevated expression of PEDS1 in immunochemotherapy-resistant tissues of esophageal squamous cell carcinoma In the aforementioned discovery phase, the detection values ​​of each protein only represent the average value in 3 samples, which may be accidental. Therefore, to verify the nature of the elevated expression of PEDS1 in immunochemotherapy-resistant tissues found in the discovery phase, this study further tested 118 resistant (treatment response rate score TGR3, 4, 5) and 70 sensitive (treatment response rate score TGR1, 2) samples using the commonly used clinical immunohistochemistry technique. The results are as follows: Figures 4-6The results showed that this experiment further confirmed the high expression of PEDS1 in ESCC tissues resistant to immunotherapy and chemotherapy in both the training and validation sets. Therefore, PEDS1 possesses the basic properties of a marker for immunotherapy and chemotherapy resistance in ESCC.

[0016] For the immunohistochemical procedures and result interpretation methods in this example, see 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.

[0017] Example 3: AUC value of PEDS1 protein expression level for identifying resistance to neoadjuvant immunotherapy in locally advanced esophageal squamous cell carcinoma The AUC value is a scientific method recognized in the field of diagnosis to measure the accuracy of markers. To measure the accuracy of PEDS1 in diagnosing resistance to neoadjuvant immunotherapy in locally advanced esophageal squamous cell carcinoma, this study plotted an ROC curve using the data from Example 2. The AUC values ​​in the training and validation sets were 0.8804 and 0.8834, respectively ( Figure 7 , 8). The data also indicate that when the threshold for immunohistochemical PEDS1 protein expression in tissue is set at 5.7, patients with an expression level above this threshold can be diagnosed as neoadjuvant immunotherapy-resistant ESCC with a sensitivity of 72.86% and a specificity of 86.44%. In summary, PEDS1 can be used as a biomarker for diagnosing immunotherapy-resistant ESCC, and PEDS1 detection reagents have application in the development of diagnostic reagents for drug resistance in locally advanced ESCC.

[0018] The above embodiments are merely preferred embodiments for the purpose of fully illustrating 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 within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. Use of a reagent for detecting PEDS1 in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in locally advanced esophageal squamous cell carcinoma, characterized by: The patient's resistance to a neoadjuvant immunochemotherapy regimen comprising an immune checkpoint inhibitor and chemotherapy drugs is determined by detecting the expression level of PEDS1 protein in esophageal squamous cell carcinoma tissue, wherein the stage of the locally advanced esophageal squamous cell carcinoma is cT3-4aN0 / +M0 or cT1b-2N+M0.

2. Use of the reagent for detecting PEDS1 according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in locally advanced esophageal squamous cell carcinoma, characterized in that: The test object is a tissue sample of esophageal squamous cell carcinoma surgically removed after neoadjuvant immunochemotherapy.

3. Use of the reagent for detecting PEDS1 according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in locally advanced esophageal squamous cell carcinoma, characterized in that: The ID of the PEDS1 in the NCBI database is 387521, and the ID in the Uniprot database is A5PLL7.

4. Use of the reagent for detecting PEDS1 according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in locally advanced esophageal squamous cell carcinoma, characterized in that: The reagent for detecting PEDS1 adopts enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, immunocoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunoassay, fluorescence immunochromatography, surface plasmon resonance, immuno-PCR or biotin avidin technique.

5. Use of the reagent for detecting PEDS1 according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in locally advanced esophageal squamous cell carcinoma, characterized in that: The detection adopts immunohistochemistry detection method.

6. Use of the reagent for detecting PEDS1 according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in locally advanced esophageal squamous cell carcinoma, characterized in that: When the QRSL1 protein expression level in immunohistochemistry was ≥5.7, the patient was judged to have drug resistance.

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