Application of MELTF detection reagent in preparation of diagnostic reagent for neoadjuvant immunochemotherapy resistance of local advanced esophageal squamous carcinoma

Abstract

The invention discloses application of a reagent for detecting MELTF 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 MELTF 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.

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

[0005] This study, through high-precision spatial proteomics and subsequent research, discovered that melanotransferrin (MELTF) possesses the potential to serve as a diagnostic marker for immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma. Therefore, a MELTF detection reagent can be used to prepare a diagnostic reagent for drug resistance in locally advanced esophageal squamous cell carcinoma. This study provides a new strategy for the development of drug resistance diagnosis and treatment methods for locally advanced esophageal squamous cell carcinoma. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a reagent for detecting MELTF for use in preparing a diagnostic reagent for neoadjuvant immunotherapy resistance in locally advanced esophageal squamous cell carcinoma.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: 1. Use of a reagent for detecting MELTF in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in locally advanced esophageal squamous cell carcinoma, wherein the neoadjuvant immunotherapy resistance in locally advanced esophageal squamous cell carcinoma refers to the resistance of primary locally advanced esophageal squamous cell carcinoma cells to immune checkpoint inhibitors combined with chemotherapy drugs.

[0008] Preferably, the locally advanced esophageal squamous cell carcinoma of the present invention refers to primary locally advanced esophageal squamous cell carcinoma, which refers to esophageal squamous cell carcinoma with a stage of cT3-4aN0 / +M0 or cT1b-2N+M0.

[0009] Preferably, the MELTF has an ID of 4241 in the NCBI database and an ID of P08582 in the Uniprot database.

[0010] Preferably, the reagent for detecting MELTF in the present invention is used to detect the expression level of MELTF protein.

[0011] Preferably, the reagent for detecting MELTF adopts enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, immunoprecipitation, 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.

[0012] Preferably, the method used in the MELTF detection reagent is an immunohistochemical detection method.

[0013] The beneficial effects of the present invention are as follows: the present invention provides an application of a reagent for detecting MELTF in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in locally advanced esophageal squamous cell carcinoma, and screens differentially expressed proteins in neoadjuvant immunotherapy resistance tissues of locally advanced esophageal squamous cell carcinoma through proteomics, thereby providing a new marker for the diagnosis of immunotherapy resistance in locally advanced esophageal squamous cell carcinoma, and also providing a basis for the development and future development of a diagnostic method for immunotherapy resistance in locally advanced esophageal squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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 MELTF 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 MELTF in surgically resected tissues of locally advanced esophageal squamous cell carcinoma with sensitivity and resistance to immunochemotherapy. Figure 5 To evaluate the immunohistochemical score of MELTF in a training set of surgically resected tissues from patients with immunochemotherapy-sensitive and -resistant locally advanced esophageal squamous cell carcinoma (n=188); Figure 6 To evaluate the immunohistochemical score of MELTF in a validation set of surgical resection tissues from patients with locally advanced esophageal squamous cell carcinoma who were sensitive to and resistant to immunochemotherapy (n=83). Figure 7 To investigate the accuracy of MELTF 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 MELTF as a biomarker for distinguishing the sensitivity of locally advanced esophageal squamous cell carcinoma to immunochemotherapy in a validation set (n=83). DETAILED DESCRIPTION

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

[0016] In the present invention, locally advanced esophageal squamous cell carcinoma refers to primary locally advanced esophageal squamous cell carcinoma, which is staged as cT3-4aN0 / +M0 or cT1b-2N+M0. The ID of MELTF in the NCBI database is 4241, and the ID in the Uniprot database is P08582.

[0017] 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 MELTF in drug-resistant samples increased significantly ( Figure 3 ).

[0018] 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 MELTF 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 MELTF 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 MELTF in ESCC tissues resistant to immunotherapy and chemotherapy in both the training and validation sets. Therefore, MELTF possesses the basic properties of a marker for immunotherapy and chemotherapy resistance in ESCC.

[0019] 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 MELTF 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.

[0020] Example 3: AUC value of MELTF 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 MELTF 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 0.8985 and 0.8939, respectively. Figure 7 , 8). Based on the data, it can be concluded that when the threshold for MELTF protein expression in tissues by immunohistochemistry is set at 6.3, patients with neoadjuvant immunotherapy-resistant esophageal squamous cell carcinoma (ESCC) can be diagnosed with a sensitivity of 85.71% and a specificity of 78.81%. In summary, MELTF can be used as a marker for diagnosing ESCC with immunotherapy-resistant ESCC, and the MELTF detection reagent has application in the development of a diagnostic reagent for drug resistance in locally advanced ESCC.

[0021] 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 MELTF in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in locally advanced esophageal squamous cell carcinoma, characterized by: The neoadjuvant immunochemotherapy resistance of locally advanced esophageal squamous cell carcinoma refers to the resistance of primary locally advanced esophageal squamous cell carcinoma cells to the combination of immune checkpoint inhibitors and chemotherapy drugs.

2. Use of the reagent for detecting MELTF 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 locally advanced esophageal squamous cell carcinoma refers to primary locally advanced esophageal squamous cell carcinoma, which refers to esophageal squamous cell carcinoma with a stage of cT3-4aN0 / +M0 or cT1b-2N+M0.

3. Use of the reagent for detecting MELTF according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in advanced esophageal squamous cell carcinoma, characterized in that: The ID of the MELTF in the NCBI database is 4241, and the ID in the Uniprot database is P08582.

4. Use of the reagent for detecting MELTF according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in advanced esophageal squamous cell carcinoma, characterized in that: The reagent for detecting MELTF is used to detect the expression level of MELTF protein.

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

6. Use of the reagent for detecting MELTF according to claim 1 in the preparation of a diagnostic reagent for neoadjuvant immunotherapy resistance in advanced esophageal squamous cell carcinoma, characterized in that: The method used by the reagent for detecting MELTF is an immunohistochemical detection method.

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