Application of RNA editing enzyme ADAR1 in predicting the effect of esophageal squamous cell carcinoma immunotherapy

Abstract

The application discloses application of an RNA editing enzyme ADAR1 in prediction of esophageal squamous cell carcinoma immunotherapy effect, and belongs to the technical field of immunotherapy.The technical problem to be solved by the application is how to predict the esophageal squamous cell carcinoma immunotherapy effect.The application provides application of a biomarker or a substance for detecting the biomarker in prediction or auxiliary prediction of immunosingle-drug treatment esophageal squamous cell carcinoma efficacy or preparation of a product for predicting or auxiliary prediction of immunosingle-drug treatment esophageal squamous cell carcinoma efficacy, and the biomarker is ADAR1 protein.Experiments prove that ADAR1 may have the potential to predict the immunosingle-drug treatment efficacy of esophageal squamous cell carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy technology, and in particular relates to the application of the RNA editing enzyme ADAR1 in predicting the efficacy of immunotherapy for esophageal squamous cell carcinoma. Background Technology

[0002] Esophageal squamous cell carcinoma is the most common pathological type of esophageal cancer in my country. Because most patients are diagnosed at an advanced stage, treatment options are limited, resulting in a high mortality rate. Improving patient survival rates and treatment options is urgently needed. In recent years, immune checkpoint inhibitors (ICIs) have represented a breakthrough in the treatment of esophageal squamous cell carcinoma. For example, monoclonal antibodies targeting PD-1 or PD-L1 can relieve T-cell immunosuppressive signals and exert anti-tumor effects. Clinical trials and real-world studies have shown that adjuvant or neoadjuvant immunotherapy can provide long-term survival benefits for patients with esophageal squamous cell carcinoma, but only a portion of patients respond to immunotherapy. Therefore, reliable biomarkers are urgently needed to screen for suitable candidates for immunotherapy.

[0003] Studies have found that the efficacy of immunotherapy for esophageal cancer is related to CD8 levels in the tumor immune microenvironment. + The number of T cells is correlated with the expression level of PD-L1 on the surface of tumor cells. This is due to the presence of a large number of CD8+ cells. + Tumor immune environments characterized by T-cell infiltration and high PD-L1 expression are associated with better immunotherapy efficacy. Currently, many promising biomarkers for predicting immunotherapy efficacy have emerged, such as PD-L1, a protein associated with the tumor inflammatory microenvironment, and tumor mutation burden (TMB), a DNA level representing neoantigens. However, these methods still have limitations. PD-L1 expression level is the only predictive biomarker approved by the US FDA. However, PD-L1 has limited sensitivity and specificity. The detection of PD-L1 expression levels is influenced by various factors, including the variety of antibody types and inconsistent cut-off values, leading to inaccuracies in PD-L1 quantification. Therefore, using this single indicator alone cannot fully reflect the immune microenvironment. At the gene level, TMB can also reflect neoantigens produced by somatic cells and tumor immunogenicity to some extent, thus predicting patient responsiveness to immunotherapy. However, relying solely on TMB cannot fully characterize the process of antigen production, presentation, and immune response, and it cannot distinguish between treatment-sensitive and insensitive individuals.

[0004] ADAR (Adenosine deaminase action RNA) is an enzyme involved in adenosine-inosine RNA editing (A-to-I RNA editing). In vertebrates, the ADAR family consists of three members: ADAR (ADAR1), ADARRB1 (ADAR2), and ADARRB2 (ADAR3). The two isoforms of ADAR1, p150 and p110, are generated using separate promoters and alternating splicing. ADAR1-p150 is primarily located in the cytoplasm, while ADAR1-p110 is primarily located in the nucleus. ADAR1p150 regulates dsRNA sensing mechanisms mediated by mitochondrial antiviral signaling protein (MAVS), melanoma differentiation-associated protein 5 (MDA5), and interferon signaling (MDA5-MAVS-ifn signaling). Summary of the Invention

[0005] The technical problem to be solved by this invention is: how to predict the efficacy of immunotherapy for esophageal squamous cell carcinoma.

[0006] To address the aforementioned technical problems, in a first aspect, the present invention provides the application of a biomarker or a substance for detecting the biomarker in P1) or P2):

[0007] P1) Predicting or assisting in predicting the efficacy of immunotherapy monotherapy for esophageal squamous cell carcinoma;

[0008] P2) Application in the preparation of products that predict or assist in predicting the efficacy of immunotherapy monotherapy for esophageal squamous cell carcinoma;

[0009] The biomarker is the ADAR1 protein.

[0010] Furthermore, in the aforementioned applications, the ADAR1 protein includes the ADAR1-p150 subtype.

[0011] Furthermore, in the aforementioned application, the substance used to detect the biomarker is a reagent for detecting the expression level and / or content of the ADAR1 protein or ADAR1-p150 subtype.

[0012] Specifically, the protein expression level refers to the abundance of the gene-encoded protein detected at the translational level.

[0013] In the above applications, the substances used to detect the biomarkers include reagents for detecting the biomarkers by reverse transcription-polymerase chain reaction, real-time quantitative PCR, transcriptome sequencing, Northern blot, in situ hybridization, gene chip technology, Nanopore sequencing, PacBio sequencing, Western blotting, immunohistochemistry, immunofluorescence, radioimmunoassay, immunoprecipitation, enzyme-linked immunosorbent assay, enzyme immunoassay, flow cytometry, high-performance liquid chromatography, capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance, immuno-PCR, or biotin-avidin technology.

[0014] In embodiments of the present invention, the expression level of the ADAR1 protein or ADAR1-p150 subtype is specifically detected by immunohistochemistry to detect the level of ADAR1 or ADAR1-p150 subtype in tumor tissue.

[0015] Furthermore, in the aforementioned application, the substance used to detect the biomarker is an antibody that specifically binds to ADAR1 or / and the ADAR1-p150 subtype.

[0016] Furthermore, in the aforementioned application, the substance used to detect the biomarker is an ADAR1 antibody or / and an ADAR1-p150 antibody.

[0017] Furthermore, in the aforementioned applications, the evaluation of predicting or assisting in predicting the efficacy of immunotherapy monotherapy for esophageal squamous cell carcinoma includes one or more of the following:

[0018] A1) Tumor microenvironment detection before and after immunotherapy for esophageal squamous cell carcinoma;

[0019] A2) Efficacy evaluation indicators for immunotherapy monotherapy of esophageal squamous cell carcinoma;

[0020] A3) Progression-free survival and / or progression-free survival rate after immunotherapy monotherapy for esophageal squamous cell carcinoma.

[0021] Furthermore, in the aforementioned application, the tumor microenvironment detection in A1) includes:

[0022] A1-1), CD8+ in tumor tissues / cells after immunotherapy for esophageal squamous cell carcinoma + Percentage of T cell infiltration;

[0023] A1-2) The expression level of PD-L1 in tumor tissues / cells after immunotherapy for esophageal squamous cell carcinoma.

[0024] Furthermore, in the aforementioned application, the immune monotherapy is performed using a PD-1 inhibitor.

[0025] Furthermore, in the aforementioned application, the immune monotherapy involves using a PD-1 inhibitor as the sole treatment drug, without employing other medications.

[0026] Furthermore, in the aforementioned applications, P2) the product is a reagent or kit for diagnosis and / or prognosis and / or prediction and / or efficacy.

[0027] The kit may be a protein immunoassay kit; the protein immunoassay kit includes specific antibodies against ADAR1 or / and ADAR1-p150 proteins.

[0028] Furthermore, in the aforementioned applications, the biomarkers are used for diagnosis and / or prognosis and / or prediction and / or efficacy evaluation.

[0029] In this invention, ADAR1 is RNA-specific adenosine deaminase 1, and its amino acid sequence is numbered NP_001102.3 in GenBank.

[0030] The ADAR1-p150 is a subtype of ADAR1, and its amino acid sequence is assigned the Genbank number NP_001020278.1.

[0031] In the above applications, the products may include reagent kits, gene chips, protein chips, immunochromatographic diagnostic strips, high-throughput sequencing platforms, or biosensors.

[0032] In the above applications, the test sample for the product can be a tumor tissue / cell sample.

[0033] Furthermore, in the above applications, the tumor tissue can be fresh or frozen tissue or formalin-fixed paraffin-embedded (FFPE) pathological tissue sections.

[0034] In this invention, the ADAR1 antibody was purchased from Abcam, catalog number ab88574.

[0035] In this invention, the ADAR1-p150 antibody was purchased from Abcam, catalog number ab126745.

[0036] In this invention, the immunotherapy can be a PD-1 immunosuppressant or a PD-L1 immunosuppressant. The PD-1 immunosuppressant can be a PD-1 antibody. The PD-1 antibody is selected from: nivolumab, pembrolizumab, sintilimab, camrelizumab, or toripalimab. In this invention, the efficacy evaluation indicators after immunotherapy monotherapy for esophageal squamous cell carcinoma are assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) based on the patient's efficacy evaluation during treatment. Efficacy evaluation indicators include complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD).

[0037] In this invention, progression-free survival refers to the time from random occurrence to the first occurrence of disease progression or death from any cause.

[0038] In this invention, progression-free survival refers to the time from random occurrence to the first occurrence of disease progression or death from any cause.

[0039] This study used immunohistochemistry to detect the expression levels of ADAR1 and the ADAR1-p150 subtype in tumor tissues from esophageal squamous cell carcinoma patients. We found that the expression levels of ADAR1 and the ADAR1-p150 subtype were significantly correlated with progression-free survival (PFS) in esophageal squamous cell carcinoma patients treated with intracellular immunoassays (ICIs). We proposed using ADAR1 and its mediated A-to-I RNA editing as biomarkers to predict the efficacy of immunotherapy, thus addressing the limitations of current biomarkers.

[0040] In this invention, the expression levels of ADAR1 and ADAR1-p150 in the tumor tissue of patients with esophageal squamous cell carcinoma are detected to predict or assist in predicting the efficacy of immunotherapy monotherapy in these patients. The judgment criteria are as follows:

[0041] In patients with low ADAR1 or ADAR1-p150 expression, immunotherapy monotherapy was more effective than [previous treatment].

[0042] Patients to be tested in the ADAR1 high expression group;

[0043] The efficacy of immunotherapy monotherapy is reflected in progression-free survival rate or progression-free survival time; at the same follow-up time, the progression-free survival rate of the test patients in the high expression group was lower than that of the test patients in the low expression group.

[0044] The experiments of this invention demonstrate that ADAR1 has the potential to predict the efficacy of immunotherapy monotherapy for esophageal squamous cell carcinoma, and that knocking down ADAR1 and ADAR1-p150 subtypes or inhibiting the expression of ADAR1 and ADAR1-p150 subtypes may enhance the antitumor effect of immunotherapy by relieving the body's immunosuppressive state. Attached Figure Description

[0045] Figure 1 ADAR1 expression is associated with the efficacy of immunotherapy for esophageal squamous cell carcinoma, among which Figure 1 In the middle section, A represents the ADAR1 expression level in patients with PR, SD, and PD. Figure 1 In the B-means, ADAR1 expression level was used to analyze the progression-free survival of esophageal squamous cell carcinoma patients who received immunotherapy. Figure 1 C represents the ADAR1 high and low expression groups and CD8 expression groups. + Percentage of T cell infiltration; Figure 1 D represents the PD-L1 level in the ADAR1 high and low expression groups (* indicates a significant difference (p < 0.05), **** indicates a significant difference (p < 0.0001));

[0046] Figure 2 The expression of the ADAR1-p150 subtype is associated with the efficacy of immunotherapy for esophageal squamous cell carcinoma, among which Figure 2 In the middle section, A represents the ADAR1 expression level in patients with PR, SD, and PD. Figure 2 In the B-means, ADAR1 expression level was used to analyze the progression-free survival of esophageal squamous cell carcinoma patients who received immunotherapy. Figure 2 C represents the ADAR1 high and low expression groups and CD8 expression groups. + Percentage of T cell infiltration; Figure 2 D represents the PD-L1 level in the high and low ADAR1 expression groups. (* indicates a significant difference (p < 0.05), ** indicates a significant difference (p < 0.05), *** indicates a significant difference (p < 0.01)).

[0047] Figure 3 The expression level of ADAR1 protein is associated with the tumor microenvironment of esophageal squamous cell carcinoma. Figure 3 In the middle group, A represents patients with low ADAR1 expression and CD8+. + The proportion of positive T-cell infiltration was significantly higher. Figure 3 The proportion of PD-L1 positive cases was significantly higher in group B. Figure 3 The proportion of C being adaptive immune tolerance is significantly higher.

[0048] Figure 4The expression level of the ADAR1-p150 subtype protein is associated with the tumor microenvironment of esophageal squamous cell carcinoma. Figure 4 Patient A in the ADAR1-p150 subtype low expression group has CD8 + The proportion of positive T-cell infiltration was significantly higher. Figure 4 The proportion of PD-L1 positive cases was significantly higher in group B. Figure 4 The proportion of C being adaptive immune tolerance is significantly higher. Detailed Implementation

[0049] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0053] Example 1: Application of ADAR1 in predicting the efficacy of immunotherapy monotherapy for esophageal squamous cell carcinoma

[0054] Case selection in this study: This is a retrospective, non-interventional clinical study. This study has been approved by the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences (Approval No.: 20 / 453-2649), conducted in accordance with the principles of the Declaration of Helsinki, and informed consent has been obtained from all participants.

[0055] This study primarily included patients with esophageal squamous cell carcinoma who received anti-PD-1 antibody therapy at the Cancer Hospital of the Chinese Academy of Medical Sciences between May 2016 and August 2019.

[0056] Inclusion criteria were: 1) patients with clinically diagnosed advanced or late-stage esophageal squamous cell carcinoma; 2) patients receiving immunotherapy for the first time, regardless of the number of lines of treatment. Exclusion criteria were: 1) esophageal adenocarcinoma, mediastinal tumors, or other types of thoracic tumors; 2) patients without baseline treatment. Enrolled patients received one of the following anti-PD-1 antibodies every two / three weeks (camrelizumab 200 mg / dose, every two weeks). Typically, patients underwent enhanced CT scans of the neck, chest, and abdomen every six weeks to assess treatment efficacy, and enhanced MRI of the head was added as needed to further evaluate the treatment effect.

[0057] This study included patients' general information such as age at initial diagnosis, sex, ECOGPS score, smoking status, pathological type, and gene mutation type.

[0058] The efficacy of treatment during the patient's treatment was assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1). The efficacy evaluation indicators included complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD).

[0059] All the above data were obtained through reviewing inpatient medical records and telephone follow-ups. The last follow-up date for this study was March 14, 2022.

[0060] This study collected surgical tissue specimens from patients prior to immunotherapy. Specimens were immediately harvested according to standard procedures and embedded in paraffin blocks after ex vivo. All surgical tissue specimens were pathologically confirmed as esophageal squamous cell carcinoma. The specimen acquisition and handling procedures were approved by the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences. Informed consent was given by all specimen providers.

[0061] This study included 15 patients with esophageal squamous cell carcinoma who underwent immunotherapy. Baseline data are detailed in Table 1.

[0062] Table 1 shows the baseline data of patients receiving immunotherapy for esophageal squamous cell carcinoma.

[0063]

[0064] Note: The numbers in the second column of Table 1 represent quantities, and the numbers in parentheses represent percentages.

[0065] 1. Detection of ADAR1 protein expression levels in tissues of 15 patients with esophageal squamous cell carcinoma

[0066] Using Human anti-ADAR1 monoclonal antibody (Abcam product, Cat#)

[0067] (ab88574) Immunohistochemistry (IHC) was used to detect the expression of ADAR1 protein in FFPE specimens of surgical tissues from 15 patients with esophageal squamous cell carcinoma. The concentration of ADAR1 antibody used was 1:200. The expression level was represented by ADAR1 staining score, with higher scores indicating higher expression levels.

[0068] ADAR1 expression was scored according to the following principle: Staining score (ADAR1 staining score) = staining intensity × percentage of positive tumor cells × 100. The staining intensity score was: no staining = 0 (negative), light yellow = 1 (weakly positive), yellow = 2 (moderately positive), and brownish-yellow = 3 (strongly positive). The percentage of positive tumor cells was calculated by examining ten randomly selected fields of view under a high-power microscope (×400). The percentage of positive tumor cells in each field was calculated as the percentage of all tumor cells in that field, and the average of the percentages from the ten fields was used as the positive tumor cell percentage.

[0069] The results are as follows Figure 1 As shown in Table 2, ADAR1 expression was significantly higher in patients in the disease progression group. Figure 1 (A)

[0070] Table 2 shows the ADAR1 staining scores and progression-free survival in tissues of 15 patients with esophageal squamous cell carcinoma.

[0071] Patient number Therapeutic effect evaluation Progress status Progress-free survival time (months) ADAR1 staining score Pt-1 SD 0 54.30 6 Pt-2 PR 1 42.87 3 Pt-3 SD 1 3.93 6 Pt-4 PR 1 8.33 4 Pt-5 PR 1 9.37 6 Pt-6 PD 1 0.67 9 Pt-7 PR 1 23.17 2 Pt-8 PD 1 1.83 12 Pt-9 PR 1 4.20 4 Pt-10 PD 1 0.87 9 Pt-11 PR 1 7.70 8 Pt-12 PD 1 0.97 12 Pt-13 PR 0 13.57 6 Pt-14 PR 0 26.83 2 Pt-15 PR 0 38.50 4

[0072] In the table above, a 1 in the progress status column 3 indicates a relapse within the follow-up period in column 4, while a 0 indicates no relapse or loss to follow-up within the follow-up period in column 4.

[0073] 2. Survival Curve

[0074] Based on the optimal cutoff value (ADAR1 staining score of 6), enrolled patients were divided into a high baseline ADAR1 expression level group (above 6) and a low baseline ADAR1 expression level group (below or equal to 6).

[0075] Survival curves were plotted based on progression status and progression-free survival time, and the results are as follows: Figure 1 As shown in Figure B, the analysis results indicate that, under the same follow-up time, the progression-free survival rate was significantly increased in the group with low ADAR1 expression levels (p < 0.001).

[0076] 3. The relationship between ADAR1 expression and the tumor immune microenvironment

[0077] CD8 in the group with low baseline ADAR1 expression levels + The proportion of T cell infiltration (CD8(ZA-0508)) and the expression level of PD-L1 (CST,#13684) were higher. Figure 1 C and Figure 1 (D).

[0078] Therefore, the expression level of ADAR1 in the tumor tissue of patients with esophageal squamous cell carcinoma can be used to predict or assist in predicting the efficacy of immunotherapy monotherapy in these patients. The criteria for judgment are as follows:

[0079] Immunotherapy in the ADAR1 low expression group was more effective or better than that in the ADAR1 high expression group.

[0080] The efficacy of immunotherapy monotherapy is reflected in progression-free survival rate or progression-free survival time; at the same follow-up time, the progression-free survival rate of the test patients in the high expression group was less than or less than that of the test patients in the low expression group.

[0081] The expression level of ADAR1 in the aforementioned tumor tissues was reflected by staining scores.

[0082] Example 2: Application of ADAR1-p150 in predicting the efficacy of immunotherapy monotherapy for esophageal squamous cell carcinoma

[0083] 1. Detection of ADAR1-p150 protein expression levels in tissues of 15 patients with esophageal squamous cell carcinoma

[0084] The expression of ADAR1-p150 protein in FFPE specimens from surgical tissues of 15 patients with esophageal squamous cell carcinoma was detected using immunohistochemistry (IHC) with Human anti-ADAR1-p150 monoclonal antibody (Abcam product, Cat#ab126745). The concentration of ADAR1-p150 antibody used was 1:200.

[0085] ADAR1-p150 staining score indicates expression level; a higher score indicates higher expression level.

[0086] ADAR1-p150 expression was scored according to the following principle: Staining score (ADAR1 staining score) = staining intensity × percentage of positive tumor cells × 100. The staining intensity score was: no staining = 0 (negative), light yellow = 1 (weakly positive), yellow = 2 (moderately positive), and brownish-yellow = 3 (strongly positive). The percentage of positive tumor cells was calculated by examining ten randomly selected fields of view under a high-power microscope (×400). The percentage of positive tumor cells in each field was calculated as the percentage of all tumor cells in that field, consisting of weakly positive, moderately positive, and strongly positive cells. The average percentage of positive tumor cells across the ten fields was then recorded as the percentage of positive tumor cells.

[0087] The results are as follows Figure 2 As shown in Table 3, ADAR1-p150 expression was significantly higher in patients in the disease progression group. Figure 2 (A)

[0088] Table 3 shows the ADAR1-p150 staining scores and progression-free survival in tissues of 15 patients with esophageal squamous cell carcinoma.

[0089]

[0090]

[0091] In the table above, a 1 in the progress status column 3 indicates a relapse within the follow-up period in column 4, while a 0 indicates no relapse or loss to follow-up within the follow-up period in column 4.

[0092] 2. Survival Curve

[0093] Based on the optimal cutoff value (ADAR1-p150 staining score of 6), enrolled patients were divided into a high baseline ADAR1-p150 expression level group (above 6) and a low baseline ADAR1-p150 expression level group (below or equal to 6).

[0094] Survival curves were plotted based on progression status and progression-free survival time, and the results are as follows: Figure 2 As shown in Figure B, the analysis results indicate that, under the same follow-up time, the progression-free survival rate was significantly increased in the group with low ADAR1-p150 expression levels (p < 0.001).

[0095] 3. The relationship between ADAR1-p150 expression and the tumor immune microenvironment

[0096] The CD8 of the sample to be tested was detected using the method provided in Example 1. + The results showed that the CD8+ expression level was significantly lower in the group with low baseline ADAR1 expression. + The proportion of T cell infiltration and the level of PD-L1 expression were higher. Figure 2 C and Figure 2 (D).

[0097] Therefore, the expression level of ADAR1-p150 in the tumor tissue of patients with esophageal squamous cell carcinoma can be detected to predict or assist in predicting the efficacy of immunotherapy monotherapy in these patients. The criteria for judgment are as follows:

[0098] Immunotherapy was more effective in patients with low ADAR1-p150 expression than in patients with high ADAR1-p150 expression.

[0099] The efficacy of immunotherapy monotherapy is reflected in progression-free survival rate or progression-free survival time; at the same follow-up time, the progression-free survival rate of the test patients in the high expression group was lower than that of the test patients in the low expression group.

[0100] The expression level of ADAR1-p150 in the aforementioned tumor tissues was reflected by staining scores.

[0101] Therefore, the expression levels of ADAR1 and ADAR1-p150 in the tumor tissue of patients with esophageal squamous cell carcinoma can be detected to predict or assist in predicting the efficacy of immunotherapy monotherapy in these patients. The criteria for judgment are as follows:

[0102] Immunotherapy was more effective in patients in the ADAR1 or ADAR1-p150 low expression group than in patients in the ADAR1 high expression group.

[0103] The efficacy of immunotherapy monotherapy is reflected in progression-free survival rate or progression-free survival time; at the same follow-up time, the progression-free survival rate of the test patients in the high expression group was lower than that of the test patients in the low expression group.

[0104] Example 3: The effect of ADAR1 on the immune microenvironment of esophageal squamous cell carcinoma

[0105] Case selection in this study: This is a retrospective, non-interventional clinical study. This study has been approved by the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences (Approval No.: 20 / 453-2649), conducted in accordance with the principles of the Declaration of Helsinki, and informed consent has been obtained from all participants.

[0106] All human tissue samples were collected from the Cancer Hospital of the Chinese Academy of Medical Sciences. ESCC tissue and adjacent non-tumor esophageal tissue were obtained from 84 patients who underwent radical resection. All ESCC cases were pathologically confirmed. Patients with ESCC had not received anti-tumor treatment or had a history of other malignancies within three years prior to diagnosis. Clinicopathological characteristics were collected through telephone interviews and routine preoperative laboratory studies. Tumor and adjacent normal tissue obtained during surgery were rapidly frozen in liquid nitrogen within 30 minutes of resection and stored at –80°C until tissue array fabrication.

[0107] This study included 84 patients with esophageal squamous cell carcinoma. Baseline data are detailed in Table 4.

[0108] Table 4 shows the baseline data of patients receiving immunotherapy for esophageal squamous cell carcinoma.

[0109]

[0110]

[0111] Table 5 shows the staining scores of ADAR1 and ADAR1-p150 and the classification of immune microenvironment indicators in the tissues of 84 patients with esophageal squamous cell carcinoma.

[0112]

[0113]

[0114]

[0115]

[0116] In the table above, CD8 in column 2 + 1 indicates positive for T cell infiltration, and 0 indicates negative; in column 3, PD-L1 status, 1 indicates positive, and 0 indicates negative; in column 4, immune microenvironment type, 1 indicates CD8... + Positive T-cell infiltration, positive PD-L1 (adaptive immune tolerance type); 2 indicates CD8 + Negative T-cell infiltration, negative PD-L1 (immune neglect type); 3 indicates CD8 + Negative T-cell infiltration, positive PD-L1 (endogenous immune resistance); 4 indicates CD8 + Positive T-cell infiltration, negative PD-L1 (immune-tolerant type).

[0117] 1. Detection of ADAR1 protein expression levels in tissues of 84 patients with esophageal squamous cell carcinoma

[0118] Referring to the method provided in Example 1, the expression of ADAR1 protein in FFPE specimens of surgical tissues from 84 patients with esophageal squamous cell carcinoma was detected by immunohistochemistry (IHC) using Human anti-ADAR1 monoclonal antibody (Abcam product, Cat#ab88574). The concentration of ADAR1 antibody used was 1:200. The expression level was represented by ADAR1 staining score, with higher scores indicating higher expression levels.

[0119] ADAR1 expression was scored according to the following principle: Staining score (ADAR1 staining score) = staining intensity × percentage of positive tumor cells × 100. The staining intensity score was: no staining = 0 (negative), light yellow = 1 (weakly positive), yellow = 2 (moderately positive), and brownish-yellow = 3 (strongly positive). The percentage of positive tumor cells was calculated by examining ten randomly selected fields of view under a high-power microscope (×400). The percentage of positive tumor cells in each field was calculated as the percentage of all tumor cells in that field, and the average of the percentages from the ten fields was used as the positive tumor cell percentage.

[0120] CD8 is performed according to the method provided in Example 1. + T-cell infiltration detection, CD8 + The criteria for positive T-cell infiltration are: when CD8+ cells are present in the tumor parenchyma... + When the proportion of T cells in the interstitial space exceeds 20%, it is judged to be CD8. + Positive T-cell infiltration.

[0121] PD-L1 positivity was detected using the method provided in Example 1. The criteria for determining PD-L1 positivity were: PD-L1 staining (Tumor proportion score, TPS) > 1 was considered PD-L1 positive.

[0122] The results are as follows Figure 3 As shown in Table 5, patients in the ADAR1 low expression group had CD8 + The proportion of positive T-cell infiltration was significantly higher. Figure 3 In China (A), the proportion of PD-L1 positive cases was significantly higher. Figure 3 In the middle class (B), the proportion of adaptive immune tolerance was significantly higher. Figure 3 (C)

[0123] 2. Detection of ADAR1-p150 protein expression levels in tissues of 84 patients with esophageal squamous cell carcinoma

[0124] Referring to the method provided in Example 2, the expression of ADAR1-p150 protein in surgical tissues (baseline tumor tissues) of 84 patients with esophageal squamous cell carcinoma was detected by immunohistochemistry (IHC) using Human anti-ADAR1-p150 monoclonal antibody (Abcam product, Cat#ab126745). The concentration of ADAR1-p150 antibody used was 1:200. The expression level was represented by ADAR1-p150 staining score, with higher scores indicating higher expression levels.

[0125] ADAR1-p150 expression was scored according to the following principle: Staining score (ADAR1 staining score) = staining intensity × percentage of positive tumor cells × 100. The staining intensity score was: no staining = 0 (negative), light yellow = 1 (weakly positive), yellow = 2 (moderately positive), and brownish-yellow = 3 (strongly positive). The percentage of positive tumor cells was calculated by examining ten randomly selected fields of view under a high-power microscope (×400). The percentage of positive tumor cells in each field was calculated as the percentage of all tumor cells in that field, consisting of weakly positive, moderately positive, and strongly positive cells. The average percentage of positive tumor cells across the ten fields was then recorded as the percentage of positive tumor cells.

[0126] CD8 is performed according to the method provided in Example 1. + T-cell infiltration detection, CD8 + The criteria for positive T-cell infiltration are: when CD8+ cells are present in the tumor parenchyma... + When the proportion of T cells in the interstitial space exceeds 20%, it is judged to be CD8. + Positive T-cell infiltration.

[0127] PD-L1 positivity was detected using the method provided in Example 1. The criteria for determining PD-L1 positivity were: PD-L1 staining (Tumor proportion score, TPS) > 1 was considered PD-L1 positive.

[0128] The results are as follows Figure 4 As shown in Table 5, patients in the ADAR1-p150 low expression group had CD8 + The proportion of positive T-cell infiltration was significantly higher. Figure 4 In China (A), the proportion of PD-L1 positive cases was significantly higher. Figure 4 In the middle class (B), the proportion of adaptive immune tolerance was significantly higher. Figure 4 (C)

[0129] Therefore, the expression level of ADAR1-p150 in the tumor tissue of patients with esophageal squamous cell carcinoma can be detected to predict or assist in predicting the efficacy of immunotherapy monotherapy in these patients. The criteria for judgment are as follows:

[0130] Immunotherapy was more effective in patients with low ADAR1-p150 expression than in patients with high ADAR1-p150 expression.

[0131] The efficacy of immunotherapy monotherapy is reflected in the immune microenvironment of esophageal squamous cell carcinoma; at the same follow-up time, the proportion of adaptive immune tolerance in the high-expression group was significantly higher than that in the low-expression group.

[0132] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of reagents for detecting biomarkers in the preparation of products for predicting or assisting in predicting the efficacy of immunotherapy monotherapy for esophageal squamous cell carcinoma; The aforementioned immunotherapy is a treatment using a PD-1 inhibitor; The biomarker is the ADAR1-p150 subtype; The amino acid sequence of the ADAR1-p150 subtype is shown in Genbank ID NP_001020278.

1.

2. The application according to claim 1, characterized in that: The reagent used to detect the biomarker is a reagent for detecting the expression level and / or content of the ADAR1-p150 subtype.

3. The application according to claim 2, characterized in that: The reagent used to detect the biomarker is an antibody that specifically binds to the ADAR1-p150 subtype.

4. The application according to claim 3, characterized in that: The reagent used to detect the biomarker is the ADAR1-p150 antibody.

5. The application according to any one of claims 1-4, characterized in that: The evaluation of predicting or assisting in predicting the efficacy of immunotherapy monotherapy for esophageal squamous cell carcinoma includes one or more of the following: A1) Tumor microenvironment detection before and after immunotherapy for esophageal squamous cell carcinoma; A2) Efficacy evaluation indicators for immunotherapy monotherapy of esophageal squamous cell carcinoma; A3) Progression-free survival and / or progression-free survival rate after immunotherapy monotherapy for esophageal squamous cell carcinoma.

6. The application according to claim 5, characterized in that: A1) The tumor microenvironment detection includes: A1-1) Percentage of CD8+ T cell infiltration in tumor tissue / cells after immunotherapy for esophageal squamous cell carcinoma; A1-2) The expression level of PD-L1 in tumor tissues / cells after immunotherapy for esophageal squamous cell carcinoma.

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