Biomarker for predicting tumor immunotherapy effect and application thereof

Abstract

The invention discloses a biomarker for predicting tumor immunotherapy effect and application of the biomarker, and belongs to the technical field of medical technology and biological detection. The biomarker is composed of the following proteins: CXCR4 and CD4, it is found that the infiltration level of CXCR4 + CD4 + T cells is significantly related to the clinical response of tumor immunotherapy, patients can be divided into a high-expression group and a low-expression group by detecting the relative expression quantity of the CXCR4 + CD4 + T cells in tumor tissue, the patients in the high-expression group are sensitive to immunotherapy, the curative effect is significant, and the patients in the low-expression group are sensitive to immunotherapy. The total survival time and the progression-free survival time are superior to those of a low-expression group. The invention provides important technical support for prediction of curative effect of tumor immunotherapy patients and formulation of a precise treatment scheme, has the advantages of high sensitivity and high specificity, and significantly promotes clinical application of immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the fields of medical technology and biological detection technology, and in particular relates to a biomarker for predicting the effect of tumor immunotherapy and its application. Background Art

[0002] In recent years, tumor immunotherapy has made breakthrough progress, especially immune checkpoint inhibitors (such as PD-1 / PD-L1 inhibitors and CTLA-4 inhibitors) have shown significant efficacy in the treatment of various malignant tumors. However, although some patients have benefited significantly from immunotherapy, a considerable proportion of patients still have poor efficacy or no response at all. This difference is mainly attributed to the complexity and heterogeneity of the individualized tumor immune microenvironment. Therefore, identifying effective predictive biomarkers and revealing their relationship with immunotherapy response has become a key issue in improving the precision of treatment. Summary of the invention

[0003] Based on the above background, the present invention provides a detection kit for detecting CXCR4+CD4+T cells, aiming to fill the gap in the prior art.

[0004] The present invention provides a biomarker combination consisting of the following proteins: CXCR4 and CD4.

[0005] The present invention also provides a reagent for detecting the above biomarker combination, wherein the reagent is an immunohistochemical reagent.

[0006] The present invention also provides the use of the above reagent in preparing a product for predicting the efficacy of pembrolizumab on non-small cell lung cancer.

[0007] In some embodiments of the present invention, the product is a reagent or a kit.

[0008] In some embodiments of the present invention, the steps of using the product are as follows:

[0009] (1) Prepare tissue sections from non-small cell lung cancer tissue samples;

[0010] (2) immunohistochemical staining of tissue sections;

[0011] (3) Scan the immunohistochemical staining results into digital images using a microscope and imaging device;

[0012] (4) By reading the slides, the intensity of the positive staining signal and the staining area of ​​the positive signal are scored respectively to obtain the immunohistochemistry score;

[0013] The immunohistochemical score was determined as follows: blue was defined as negative staining and was defined as 0 points, light yellow was defined as weak positive staining and was defined as 1 point, brown was defined as moderate positive staining and was defined as 2 points, and dark brown was defined as strong positive staining and was defined as 3 points; when scoring the percentage of positive cells in the area of ​​the tissue, a positive cell area ratio of <25% was defined as 1 point, 25%≤positive cell area ratio<50 was defined as 2 points, 50%≤positive area ratio<75% was defined as 3 points, and a positive area ratio of ≥75% was defined as 4 points; the product of the score for staining intensity and the score for the percentage of positive cells in the area of ​​the tissue was used as the immunohistochemical score, and a score greater than or equal to 8 points was high expression, and a score less than 8 points was low expression.

[0014] In some embodiments of the present invention, when both biomarkers CXCR4 and CD4 are lowly expressed, the immunohistochemistry combined score is defined as 0 points; when one of the biomarkers CXCR4 and CD4 is lowly expressed and the other is highly expressed, the immunohistochemistry combined score is defined as 1 point; when both CXCR4 and CD4 are highly expressed, the immunohistochemistry combined score is defined as 2 points; patients with an immunohistochemistry combined score of 1-2 have a good therapeutic effect of tumor immunotherapy.

[0015] In some embodiments of the present invention, the non-small cell lung cancer tissue sample is a tumor puncture specimen before immunotherapy or a postoperative tumor tissue.

[0016] The present invention also provides a product for predicting the efficacy of pembrolizumab on non-small cell lung cancer, wherein the product contains the above-mentioned reagent.

[0017] In some embodiments of the invention, the product is a kit.

[0018] In some embodiments of the present invention, the reagent contains CXCR4 primary antibody, CD4 primary antibody, horseradish peroxidase labeled secondary antibody, 2-4% H2O2 solution, 0.5-1.5% BSA blocking solution, DAB colorimetric reagent, hematoxylin, xylene and ethanol.

[0019] CXCR4 is a G protein-coupled receptor with seven transmembrane domains that was first discovered in peripheral blood leukocytes and is highly expressed in a variety of cells, including lymphocytes, endothelial cells, epithelial cells and hematopoietic stem cells, interstitial fibroblasts and cancer cells. CXCR4 plays a key role in a variety of physiological processes, such as neurogenesis, germ cell development and angiogenesis. The basal expression of CXCR4 is mainly regulated by Nuclear Respiratory Factor 1 (NRF-1) and the nuclear transcription factor Yin-Yang 1 (YY1), the former promoting receptor transcription and the latter negatively regulating. CXCR4 responds to different signaling molecules to increase transcriptional expression, such as second messengers (calcium, cyclic AMP), cytokines (IL-2, IL-4, IL-7, IL-10, IL-15, TGF-β) and growth factors (bFGF, VEGF). However, the expression of CXCR4 is downregulated in the presence of cytokines such as TNF-a, INF-γ and IL-1β.

[0020] CXCR4, as the most commonly expressed chemokine receptor on tumor cells, is associated with the development and metastasis of a variety of tumors. Cancer cells preferentially metastasize to specific organs through the blood and lymphatic vessels, which is a huge challenge for cancer eradication. A class of GPCRs closely related to tumor metastasis is chemokine receptors. After chemokines are locally released into the microenvironment of the tumor area in an autocrine or paracrine manner, they enhance the viability and survival of cancer cells in the tumor area and environment. CXCR4 is the most widely expressed chemokine receptor in 23 human cancers, including breast cancer, ovarian cancer, melanoma, prostate cancer, and colorectal cancer, although CXCR4 is lowly expressed or not expressed in many normal tissues.

[0021] "CXCR4 plays a role in leukocyte chemotaxis in inflammatory conditions in many autoimmune diseases, and high levels of CXCR4 have been observed in different types of human cancers. These findings suggest that CXCL12 / CXCR4 are potential therapeutic targets for autoimmune diseases and open up new avenues for targeted cancer therapy by neutralizing CXCL12 and CXCR4."

[0022] At present, the research targeting CXCR4 is in progress or has become quite mature. In addition to the FDA-approved CXCR4 inhibitor Plerixafor, which is used for hematological malignancies. In recent years, with the further understanding of the functional mechanism of CXCR4, a variety of CXCR4 targeted inhibitors have been explored. For example, Motixafortide, which has been approved for clinical trials in China, is currently in clinical phase III for the treatment of locally advanced or metastatic triple-negative breast cancer; Mavorixafor, which is also in clinical phase III, acts on hematopoietic stem cell mobilization for autologous bone marrow transplantation in patients with multiple myeloma. It is planned to submit NDA in the first half of this year and is expected to become the world's second CXCR4 targeted drug. It is believed that soon, treatment methods targeting the CXCR4 receptor will provide new breakthroughs in the treatment of various diseases such as cancer.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The applicant has found through in-depth research that the expression of CXCR4 and CD4 in tumor patient tissues treated with immunotherapy can be used to determine the sensitivity of patients to immunotherapy. Based on the correlation between the expression of CXCR4 and CD4 and immunotherapy for tumor patients, the present invention uses CXCR4 and CD4 as molecular markers to detect their expression, which can be used as a predictive indicator for the efficacy of immunotherapy for tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph showing the expression levels of CXCR4 and CD4 detected by immunohistochemical staining of non-small cell lung cancer specimens in Example 1.

[0026] Figure 2It is a Kaplan-Meier survival analysis diagram of CXCR4 and CD4 expression on prognosis in Example 1. The red curve represents patients with high expression of CXCR4 CD4 (CXCR4 CD4 high), and the blue curve represents patients with low expression of CXCR4 CD4 (CXCR4 CD4low). The red curve is above the blue curve, indicating that the overall survival probability of patients with high expression is higher. Pan-cancer refers to bladder cancer, esophageal adenocarcinoma, glioblastoma, hepatocellular carcinoma, head and neck cancer, melanoma, non-small cell lung cancer, and urothelial carcinoma. Among them, A is the effect of CXCR4 and CD4 expression on the overall survival of tumor patients who have received anti-PD-1 immunotherapy (pembrolizumab), and the numbers under the bottom "Low" are 140, 64, 38, 17, 1, 1, respectively, representing the number of patients remaining in the low expression group at different time points; the numbers under "High" are 314, 211, 138, 64, 12, 7, representing the number of patients remaining in the high expression group at different time points. B is the effect of CXCR4 and CD4 expression on the overall survival of tumor patients who received anti-PD-L1 immunotherapy (durvalumab). The numbers 142, 65, 57, 45, 29, 5, 1, and 0 under "Low" at the bottom are the number of remaining patients in the low expression group at different time points; the numbers 317, 231, 179, 141, 78, 20, 7, and 1 under "High" at the bottom are the number of remaining patients in the high expression group at different time points. C is the effect of CXCR4 and CD4 expression on the overall survival of tumor patients who received anti-CTLA-4 immunotherapy (tremelimumab). The numbers 49, 29, 19, 8, 0, 0, and 1 under "Low" at the bottom are the number of remaining patients in the low expression group at different time points; the numbers 72, 53, 39, 19, 10, 5, 1, and 1 under "High" at the bottom are the number of remaining patients in the high expression group at different time points.

[0027] Figure 3 It is the Kaplan-Meier survival analysis diagram of CXCR4 and CD4 expression for prognosis in Example 2. The black line represents the overall survival (OS) curve of patients in the low expression group of CXCR4 CD4, and the red line represents the overall survival curve of patients in the high expression group of CXCR4 CD4. By comparing these two curves, it can be intuitively seen that the survival differences of patients with different expression levels when receiving different immunotherapy. The numbers at the bottom of the chart represent the number of patients in the corresponding expression level group (high expression or low expression) at different time points. Pan-cancer refers to bladder cancer, esophageal adenocarcinoma, glioblastoma, hepatocellular carcinoma, head and neck cancer, melanoma, non-small cell lung cancer, and urothelial carcinoma. Wherein, A is the effect of CXCR4 and CD4 expression on the overall survival of tumor patients who have received different immunotherapy, and BC is the effect of CXCR4 and CD4 expression on the progression-free survival of tumor patients who have received different immunotherapy.

[0028] Figure 4 The ROC curve in Example 2 is used to evaluate the diagnostic efficacy of the kit for detecting CXCR4 and CD4 levels to predict the efficacy of tumor immunotherapy.

[0029] Figure 5 The ROC curve in Example 2 is used to support that the combined detection of CXCR4 and CD4 expression levels can be used as an effective biomarker, and its predictive ability is verified by an independent patient data set. DETAILED DESCRIPTION

[0030] In the following examples, the preparation of the immunohistochemical test reagents used is as follows:

[0031] 1. Preparation of gradient ethanol solutions: 75%, 85%, and 95% ethanol solutions are prepared by diluting anhydrous ethanol and distilled water.

[0032] 2. Preparation of 1×EDTA antigen retrieval solution (pH 9.0): Take 121.1g Tris, add water to 1L, then take 186.1g EDTA, add water to 1L. Take 1mL 1mol / LTris and 2mL 500mmol / L EDTA, adjust the pH to 9.0, and add water to 1000mL.

[0033] Example 1

[0034] In this example, immunohistochemical staining of non-small cell lung cancer tissue before immunotherapy was performed, and the steps were as follows:

[0035] (1) Prepare and cut 4 μm paraffin sections of non-small cell lung cancer; bake the sections on a slide spreader at 37°C for 30 min and then place them in a 65°C oven for 1 h;

[0036] (2) Dewaxing the slices obtained in step (1), specifically: xylene I for 10 min → xylene II for 10 min → xylene III for 10 min → 100% ethanol for 5 min → 95% ethanol for 5 min → 85% ethanol for 5 min → 75% ethanol for 5 min → ddH 2 O5min;

[0037] (3) Antigen retrieval: Add the prepared TRIS-EDTA into the cylinder containing the slices, put it into the pressure cooker after boiling water, and continue for 3 minutes after the temperature rises to 120°C;

[0038] (4) Cool naturally to room temperature, ddH 2 O washing 5min×3;

[0039] (5) Step (4) was treated with ddH 2 O-washed sections were placed in 1×EDTA antigen retrieval solution and boiled for 30 min;

[0040] (6) Cool naturally to room temperature, ddH 2 O washing 5min×3;

[0041] (7) Blocking endogenous peroxidase: Add 3% hydrogen peroxide to the slices for blocking, place them flat on a wet box filled with a small amount of distilled water, incubate in a dark oven at 37°C for 8 min, and place in ddH 2 O washing 5min×3;

[0042] (8) After blocking with 1% BSA at 37°C for 30 min, shake off the blocking solution, do not wash, and directly add primary antibody for incubation (CXCR4 primary antibody (ab197203) and CD4 primary antibody (ab133616) are rabbit monoclonal antibodies, abcam, dilution ratio 1:500). Place in a humidified box at 4°C refrigerator overnight; take out from 4°C refrigerator, warm to room temperature for 15 min, wash with 0.01 mol / L PBS buffer for 5 min×4;

[0043] (9) Add secondary antibody (goat anti-rabbit IgG antibody (G1302-10ML, Sewell Company) and incubate at 37°C for 45 min;

[0044] (10) After washing with 0.01 mol / L PBS buffer for 5 min × 4, the cells were developed with DAB colorimetric reagent for 2–10 min and observed under a microscope;

[0045] (11) Use ddH 2 O washing to stop color development, and then counterstaining with hematoxylin for 10 seconds;

[0046] (12) After differentiation, tap water turns blue and is soaked in distilled water;

[0047] (13) Dehydration, transparency, and sealing: After soaking in distilled water, the slices were placed in 80% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 10 min, xylene I for 10 min, and xylene II for 10 min. After taking out, drip sealing agent into the slices and place them in a fume hood to dry.

[0048] (14) The sealed sections were placed under a microscope to observe positive staining, and three fields of view were randomly selected from the non-small cell lung cancer tissues and photographed, scored, and averaged;

[0049] (15) Semi-quantitative determination of staining results: Semi-quantitative determination was performed by comprehensively evaluating the staining intensity and the proportion of positive cells under a high-power microscope (200×).

[0050] The specific scores are as follows:

[0051] (1) In the immunohistochemical detection of non-small cell lung cancer samples, blue was defined as negative staining and was defined as 0 points, light yellow was defined as weak positive staining and was defined as 1 point, brown was defined as moderate positive staining and was defined as 2 points, and dark brown was defined as strong positive staining and was defined as 3 points. When scoring the percentage of positive cells (weakly positive cells, moderately positive cells, and strongly positive cells) in the area of ​​the tissue, a positive cell area percentage of <25% was defined as 1 point, a positive cell area percentage of 25%≤<50% was defined as 2 points, a positive area percentage of 50%≤<75% was defined as 3 points, and a positive area percentage of ≥75% was defined as 4 points. The product of the score of staining intensity and the score of the percentage of positive cells in the area of ​​the tissue was used as the immunohistochemical score. A score greater than or equal to 8 points was considered high expression, and a score less than 8 points was considered low expression.

[0052] (2) When both biomarkers CXCR4 and CD4 are lowly expressed, the combined immunohistochemical score is defined as 0 points; when one of the biomarkers CXCR4 and CD4 is lowly expressed and the other is highly expressed, the combined immunohistochemical score is defined as 1 point; when both CXCR4 and CD4 are highly expressed, the combined immunohistochemical score is defined as 2 points; patients with an immunohistochemical combined score of 1-2 have a good therapeutic effect on tumor immunotherapy.

[0053] The experimental results are as follows Figure 1 As shown, 1A represents high expression of CXCR4, 1B represents low expression of CXCR4, 1C represents high expression of CD4, and 1D represents low expression of CD4.

[0054] Figure 2 To investigate the effects of CXCR4 and CD4 expression on the overall survival of tumor patients treated with different immunotherapies.

[0055] Example 2

[0056] This example verifies the conclusion in Example 1, and the results are as follows:

[0057] Figure 3The black line represents the overall survival (OS) curve of patients with low CXCR4 CD4 expression, and the red line represents the overall survival curve of patients with high CXCR4 CD4 expression. By comparing these two curves, we can intuitively see the difference in survival of patients with different expression levels when receiving different immunotherapies. The numbers at the bottom of the chart represent the number of patients in the corresponding expression level group (high expression or low expression) at different time points. Pan-cancer refers to bladder cancer, esophageal adenocarcinoma, glioblastoma, hepatocellular carcinoma, head and neck cancer, melanoma, non-small cell lung cancer, and urothelial carcinoma. Among them, A is the effect of CXCR4 and CD4 expression on the overall survival of tumor patients who received different immunotherapies, and BC is the effect of CXCR4 and CD4 expression on the progression-free survival of tumor patients who received different immunotherapies.

[0058] Select two more batches of samples to draw the ROC curve, and the results are as follows:

[0059] Figure 4 ROC curve was used to evaluate the diagnostic efficacy of the kit for detecting CXCR4 and CD4 levels in predicting the efficacy of anti-PD-1 therapy (pembrolizumab) immunotherapy for non-small cell lung cancer. The sample size was 114 cases, of which 80 cases had immunohistochemistry scores of 1-2 and the remaining immunohistochemistry scores were all 0.

[0060] Figure 5 The ROC curve was used to support that the combined detection of CXCR4 and CD4 expression can be used as an effective biomarker, and the predictive ability of anti-PD-1 therapy (pembrolizumab) on the efficacy of immunotherapy for non-small cell lung cancer was verified through an independent patient data set. The sample size was 76 cases, of which 40 cases had immunohistochemistry scores of 1-2, and the remaining immunohistochemistry scores were all 0.

[0061] In summary, the expression levels of CXCR4 and CD4 are closely related to the efficacy of immunotherapy, and the expression levels of CXCR4 and CD4 can be used as markers to predict the efficacy of immunotherapy.

[0062] Example 3

[0063] This embodiment provides a kit for predicting tumor immunotherapy, wherein the detection reagent is used to detect CXCR4 and CD4, and the kit contains CXCR4 primary antibody, CD4 primary antibody, horseradish peroxidase labeled secondary antibody, 2-4% H 2 O 2 solution, 0.5-1.5% by volume BSA blocking solution, DAB color developing reagent, hematoxylin, xylene and ethanol.

[0064] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A biomarker combination, characterized in that: The biomarker panel consisted of the following proteins: CXCR4 and CD4.

2. A reagent for detecting the biomarker combination as claimed in claim 1, characterized in that: The reagent is an immunohistochemical reagent.

3. Use of the reagent according to claim 2 in the preparation of a product for predicting the efficacy of pembrolizumab in non-small cell lung cancer.

4. The use according to claim 3, characterized in that: The product is a reagent or a kit.

5. The use according to claim 3 or 4, characterized in that: The steps for using the product are as follows: (1) Prepare tissue sections from non-small cell lung cancer tissue samples; (2) immunohistochemical staining of tissue sections; (3) Scan the immunohistochemical staining results into digital images using a microscope and imaging device; (4) By reading the slides, the intensity of the positive staining signal and the staining area of ​​the positive signal are scored respectively to obtain the immunohistochemistry score; The immunohistochemical score was determined as follows: blue was defined as negative staining and was defined as 0 points, light yellow was defined as weak positive staining and was defined as 1 point, brown was defined as moderate positive staining and was defined as 2 points, and dark brown was defined as strong positive staining and was defined as 3 points; when scoring the percentage of positive cells in the area of ​​the tissue, a positive cell area ratio of <25% was defined as 1 point, 25%≤positive cell area ratio<50 was defined as 2 points, 50%≤positive area ratio<75% was defined as 3 points, and a positive area ratio of ≥75% was defined as 4 points; the product of the score for staining intensity and the score for the percentage of positive cells in the area of ​​the tissue was used as the immunohistochemical score, and a score greater than or equal to 8 points was high expression, and a score less than 8 points was low expression.

6. The use according to claim 5, characterized in that: When both biomarkers CXCR4 and CD4 are lowly expressed, the immunohistochemistry combined score is defined as 0 points; when one of the biomarkers CXCR4 and CD4 is lowly expressed and the other is highly expressed, the immunohistochemistry combined score is defined as 1 point; when both CXCR4 and CD4 are highly expressed, the immunohistochemistry combined score is defined as 2 points; patients with an immunohistochemistry combined score of 1-2 have a good efficacy of tumor immunotherapy.

7. The use according to claim 6, characterized in that: The non-small cell lung cancer tissue sample is a tumor puncture specimen before immunotherapy or a postoperative tumor tissue.

8. A product for predicting the efficacy of pembrolizumab in non-small cell lung cancer, characterized in that: The product contains the reagent described in claim 2.

9. The product according to claim 8, characterized in that The product is a test kit.

10. The product according to claim 9, characterized in that The reagent contains CXCR4 primary antibody, CD4 primary antibody, horseradish peroxidase labeled secondary antibody, 2-4% H2O2 solution by volume, 0.5-1.5% BSA blocking solution by volume, DAB color developing reagent, hematoxylin, xylene and ethanol.

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