Application of CDK5RAP2 protein in preparation of kit for diagnosing neoadjuvant immunochemotherapy resistance of local advanced esophageal squamous carcinoma

Abstract

The invention discloses an application of CDK5RAP2 protein in preparation of a kit for diagnosing neoadjuvant immunochemotherapy resistance of local advanced esophageal squamous carcinoma. Differential expression protein in neoadjuvant immunochemotherapy resistance tissues of local advanced esophageal squamous carcinoma is 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] This invention relates to the field of detection, specifically to the use of a CDK5RAP2 protein in the preparation of a kit for diagnosing neoadjuvant immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma. Background Technology

[0002] Esophageal cancer (EC) is one of the most common malignant tumors worldwide and a major threat to the health of Chinese residents. Although neoadjuvant chemoradiotherapy (NCRT) or neoadjuvant chemotherapy (NCT) combined with surgical resection can improve the survival rate of patients with locally advanced EC, the prognosis for some patients remains unsatisfactory. In recent years, PD-1 inhibitors, by blocking the interaction between PD-1 and PD-L1, can restore the anti-tumor activity of T lymphocytes, enhance the immune response, and reduce tumor cell proliferation and metastasis, and have become a very popular cancer immunotherapy. Immunochemotherapy (ICT) has become the standard first-line treatment for advanced EC, bringing significant survival benefits to patients. Whether neoadjuvant immunochemotherapy (NICT) can bring more cure opportunities to patients with locally advanced EC has become a key research direction. Currently, several clinical studies on neoadjuvant immunotherapy for EC are underway and have achieved surprising results. Sintilimab is a fully humanized recombinant IgG4 monoclonal antibody targeting PD-1. It offers clinical advantages such as relatively low treatment cost and is considered safe and effective in a clinical setting. Although multiple studies have confirmed the clinical efficacy of neoadjuvant sintilimab in treating EC, some patients still fail to benefit from NCT, and may even experience increased risk of disease progression, treatment-related adverse reactions, increased surgical difficulty, or increased risk of postoperative complications.

[0003] Establishing effective diagnostic methods for NCT resistance in locally advanced esophageal squamous cell carcinoma is crucial for improving the diagnosis and treatment of esophageal squamous cell carcinoma, 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 CDK5 regulatory subunit associated protein 2 (CDK5RAP2) possesses the property of serving as a biomarker for diagnosing drug resistance to immunochemotherapy in locally advanced esophageal squamous cell carcinoma. Therefore, CDK5RAP2 detection reagents can be used to prepare diagnostic reagents 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. 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 CDK5RAP2 in the preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma.

[0007] To achieve the above objectives, the present invention provides the following technical solution: 1. The use of a CDK5RAP2 protein in the preparation of a kit for diagnosing neoadjuvant immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma, the kit comprising a reagent for detecting the expression level of CDK5RAP2 protein; the resistance is diagnosed by detecting the expression level of CDK5RAP2 protein in esophageal squamous cell carcinoma tissue samples, and high expression of CDK5RAP2 protein indicates neoadjuvant immunochemotherapy resistance.

[0008] Preferably, the esophageal squamous cell carcinoma tissue sample is a surgically removed tissue sample after neoadjuvant immunochemotherapy.

[0009] Preferably, the expression level of the CDK5RAP2 protein in this invention is determined by enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay, immunoprecipitation assay, Western blotting, 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. Preferably, the expression level of the CDK5RAP2 protein is detected by immunohistochemistry.

[0010] Preferably, in this invention, when the expression level score of CDK5RAP2 protein detected by immunohistochemistry is higher than 5.7, it is judged to be drug-resistant esophageal squamous cell carcinoma.

[0011] Preferably, the neoadjuvant immunochemotherapy of the present invention comprises a PD-1 inhibitor combined with platinum-based and taxane-based chemotherapy drugs, wherein the PD-1 inhibitor is a monoclonal antibody.

[0012] The beneficial effects of this invention are as follows: This invention provides the application of a reagent for detecting CDK5RAP2 in the preparation of a diagnostic reagent for neoadjuvant immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma. By screening differentially expressed proteins in neoadjuvant immunochemotherapy resistant tissues of locally advanced esophageal squamous cell carcinoma through proteomics, it provides a new biomarker for the diagnosis of immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma, and also provides a foundation for the development and future advancement of diagnostic methods for immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma. Attached Figure Description

[0013] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 Spatial proteomics was used to classify esophageal squamous cell carcinoma cases into chemotherapy-resistant and immune checkpoint inhibitor-sensitive groups based on tumor regression grade. Figure 2 Differences in protein profiles of surgically resected tissues from locally advanced esophageal squamous cell carcinoma cases that were sensitive to and resistant to immunochemotherapy. Figure 3 The relative mass spectrometry values ​​of CDK5RAP2 in surgically resected tissues of locally advanced esophageal squamous cell carcinoma cases that were sensitive and resistant to neoadjuvant immunochemotherapy. Figure 4 Immunohistochemical staining images of representative samples of CDK5RAP2 in surgically resected tissues from locally advanced esophageal squamous cell carcinoma cases that were sensitive and resistant to immunochemotherapy. Figure 5 Immunohistochemical score of CDK5RAP2 in a training set of surgically resected tissues (n=188) of locally advanced esophageal squamous cell carcinoma cases that were sensitive and resistant to immunochemotherapy; Figure 6 Immunohistochemical score of CDK5RAP2 in a validation set of surgically resected tissues (n=83) of locally advanced esophageal squamous cell carcinoma cases that were sensitive and resistant to immunochemotherapy; Figure 7 To investigate the accuracy of CDK5RAP2 as a biomarker in differentiating the sensitivity of locally advanced esophageal squamous cell carcinoma to immunochemotherapy in the training set (n=188); Figure 8 To investigate the accuracy of CDK5RAP2 as a biomarker in differentiating the sensitivity of locally advanced esophageal squamous cell carcinoma to immunochemotherapy in a validation set (n=83). Detailed Implementation

[0014] 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.

[0015] Example 1: Screening for biomarkers of immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma using proteomics analysis strategies. The main drugs used in immunochemotherapy for locally advanced esophageal squamous cell carcinoma 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 esophageal squamous cell carcinoma. 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 CDK5RAP2 was significantly increased in the drug-resistant samples ( Figure 3 ).

[0016] The method for obtaining esophageal squamous cell carcinoma tissue samples after immunochemotherapy and the method for detecting them using high-precision spatial proteomics technology in this embodiment 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, LisaSchweizer, 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, 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 CDK5RAP2 expression in esophageal squamous cell carcinoma tissue 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 elevated expression of CDK5RAP2 in immunochemotherapy-resistant tissues discovered in the discovery phase, this study further examined 118 resistant samples (treatment response rate scores TGR3, 4, and 5) and 70 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 CDK5RAP2 in esophageal squamous cell carcinoma tissues resistant to immunochemotherapy in both the training and validation sets. Therefore, CDK5RAP2 possesses the fundamental properties to serve as a biomarker for immunochemotherapy resistance in esophageal squamous cell carcinoma.

[0017] 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 CDK5RAP2 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.

[0018] Example 3: AUC value of CDK5RAP2 protein expression level in differentiating neoadjuvant immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma. AUC (Acceptance Value of Criteria) is a scientifically recognized method for measuring the accuracy of biomarkers in the diagnostic field. To measure the accuracy of CDK5RAP2 in diagnosing neoadjuvant immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma, this study plotted ROC curves using data from Example 2. The AUC values ​​in the training and validation sets were 0.8737 and 0.8400, respectively. Figure 7 , Figure 8 Based on the data, it can be concluded that when the threshold is set at 5.7 for CDK5RAP2 protein expression in tissues detected by immunohistochemistry, patients with esophageal squamous cell carcinoma above this threshold can be diagnosed with neoadjuvant immunochemotherapy-resistant esophageal squamous cell carcinoma. The sensitivity of this diagnosis is 82.86%, and the specificity is 83.05%. In conclusion, CDK5RAP2 can be used as a biomarker for diagnosing immunochemotherapy-resistant esophageal squamous cell carcinoma, and CDK5RAP2 detection reagents have applications in preparing diagnostic reagents for drug resistance in locally advanced esophageal squamous cell carcinoma.

[0019] 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. The use of CDK5RAP2 protein in the preparation of a kit for diagnosing neoadjuvant immunochemotherapy resistance in locally advanced esophageal squamous cell carcinoma, the kit comprising reagents for detecting CDK5RAP2 protein expression levels; characterized in that: The drug resistance was diagnosed by detecting the expression level of CDK5RAP2 protein in esophageal squamous cell carcinoma tissue samples, and the high expression of CDK5RAP2 protein indicated resistance to neoadjuvant immunochemotherapy.

2. The use as described in claim 1, characterized in that: The esophageal squamous cell carcinoma tissue sample was a surgically removed tissue sample after neoadjuvant immunochemotherapy.

3. The use as described in claim 1, characterized in that: The expression level of CDK5RAP2 protein was determined using enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay, immunoprecipitation assay, Western blotting, 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. The use as described in claim 1 is characterized in that the expression level of the CDK5RAP2 protein is detected by immunohistochemistry.

4. The use as described in claim 4, characterized in that: When the CDK5RAP2 protein expression level score detected by immunohistochemistry is higher than 5.7, it is judged as drug-resistant esophageal squamous cell carcinoma.

5. The use as described in claim 1, characterized in that: The neoadjuvant immunochemotherapy comprises a PD-1 inhibitor in combination with platinum-based and taxane-based chemotherapy drugs, wherein the PD-1 inhibitor is a monoclonal antibody.

Images