A Tumor Tissue PD-L1 Gradient Quality Control Slide and Its Preparation Method and Application

Abstract

This application relates to the field of biopathological detection technology, and specifically discloses a tumor tissue PD-L1 gradient quality control slide and its preparation method and application. The tumor tissue PD-L1 gradient quality control slide of this application includes a glass slide label area, a staining control area, and a tissue attachment area. In the staining control area, three HIS-PDTX tumor living tissues or hPBMC-PDTX tumor living tissue samples with PD-L1 staining gradients serving as controls, namely PD-L1 staining gradient being negative, PD-L1 staining gradient being medium expression, and PD-L1 staining gradient being high expression, are fixed, while ensuring the traceability and continuous supply of the sample sources; the above samples can accurately reflect the distribution of PD-L1 in the HIS-PDTX tumor living tissue or hPBMC-PDTX tumor living tissue structure. By using these samples as controls, the accuracy and reliability of the tumor tissue PD-L1 gradient quality control slide can be improved.

Description

Technical Field

[0001] This application relates to the technical field of biopathological detection, and particularly relates to a tumor tissue PD-L1 gradient quality control slide and its preparation method and application. Background Art

[0002] Programmed death-ligand 1 (PD-L1) is a cell surface protein that can be detected in immune cells, including B cells, T cells, macrophages, dendritic cells (DCs), and mast cells. In addition, it is also widely expressed in cancer tissues, such as lung cancer, esophageal cancer, liver cancer, breast cancer, urothelial carcinoma, malignant melanoma, cervical cancer, renal cancer, colorectal cancer, and ovarian cancer. By binding to its receptor PD-1, PD-L1 can inhibit cellular immunity and cytokine signal transduction pathways, leading to immune tolerance and immune escape. In malignant tumors, tumor cells highly express PD-L1, bind to PD-1 on the surface of immune cells, inhibit the activity of T cells, and evade the attack of immune cells. In addition, PD-L1 can also negatively regulate immune activity by binding to other receptors (such as CD80, B7-1, B7-2, etc.).

[0003] Immunotherapy has shown effectiveness and safety in the treatment of various tumors and has been widely applied. Among them, PD-1 / PD-L1 inhibitors have become a hot topic in clinical research. Currently, 8 PD-1 / PD-L1 monoclonal antibody drugs have been approved for marketing in China, including 4 imported PD-1 / PD-L1 inhibitors and 4 domestic PD-1 inhibitors. With the approval of PD-1 / PD-L1 immune checkpoint inhibitors, cancer patient PD-L1 immunohistochemistry detection reagents, etc., have also been approved as companion diagnostics or supplementary diagnostics according to the needs of indications. At the same time, all the reagents approved by the US FDA for use in conjunction with anti-PD-1 antibody or anti-PD-L1 antibody drugs are antibody detection reagents for PD-L1 protein. The detection method is immunohistochemical detection of paraffin-embedded tissue (FFPE) samples.

[0004] Currently, the main interpretation methods for PD-L1 detection antibodies used in companion diagnostics mainly include TPS, IC, CPS, TAP, and TC combined with IC. Different types of tumors are suitable for different interpretation methods. For non-small cell lung cancer, when using the 22C3 antibody, the TC interpretation method is adopted; for triple-negative breast cancer and urothelial carcinoma, when using the SP142 antibody, the IC interpretation method is adopted; while the SP142 antibody uses a combined interpretation of TC and IC. When the FDA approved the 22C3 antibody, it stated that the TC interpretation method is used for non-small cell lung cancer, but for gastric adenocarcinoma and gastroesophageal junction adenocarcinoma, the CPS interpretation method is recommended. The CPS interpretation method is also recommended for cervical cancer and urothelial carcinoma. Therefore, accurately interpreting PD-L1 results requires the experience of professional pathologists and a large amount of training, so it is relatively difficult to promote and apply. Among them, the interpretation methods of IC, CPS, TC combined with IC, and TAP all require interpreting the PD-L1 positive staining of immune cells, and the commonly used PD-L1 staining platforms on the market mainly include the AutoStainerLink 48 platform, the Ventana Benchmark Ultra platform, and the Leica Bond Max / III platform. These platforms use different secondary antibody systems, resulting in significant differences in the staining results of the same monoclonal antibody on each platform. Considering avoiding staining differences, companion diagnostic products on the market usually do not judge the staining intensity, but mainly focus on the positive or negative interpretation of PD-L1. Such a strategy can ensure the consistency of results and minimize the variability between different platforms. However, the control samples of current PD-L1 antibody detection kits are mostly tumor cell lines or tonsil tissues. The biological characteristics of these control samples are unstable and the sources are limited. Therefore, it is difficult to trace the sample source and achieve sustainable supply, and it is impossible to perform dual PD-L1 staining interpretation on tumor cells and immune cells in tumor tissues. Moreover, when using immunohistochemistry to detect the expression of PD-L1, due to the lack of a PD-L1 standard control product, problems such as false positives and false negatives often occur in the PD-L1 immunohistochemical staining results. When using different PD-L1 antibodies, batch effects may be observed.

[0005] In summary, developing a PD-L1 gradient quality control slide for tumor tissues, which can show the PD-L1 immunohistochemical staining of tumor cells and immune cells in tumor tissues and perform comprehensive interpretation, can provide strong guidance for accurately interpreting PD-L1 results and is of great significance for the diagnosis and treatment of cancer. Summary of the Invention

[0006] The purpose of this application is to overcome the deficiencies of the above-mentioned prior art and provide a tumor tissue PD-L1 gradient quality control slide, its preparation method and application. The tumor tissue PD-L1 gradient quality control slide designed in this application can display the staining of PD-L1 in tumor cells and immune cells, provide strong guidance for accurately interpreting PD-L1 results, and has important significance for the diagnosis and treatment of cancer. Through this innovative method, it can better guide clinical treatment decisions, provide personalized treatment plans for patients, and help improve the prognosis and survival rate of cancer patients. Therefore, the development of such a quality control slide has important application prospects and significance in the field of cancer.

[0007] To achieve the above object, the technical solution adopted in this application is as follows:

[0008] This application provides a preparation method of a tumor tissue PD-L1 gradient quality control slide, including the following steps:

[0009] S1. According to the transcriptome sequencing RNA-seq information characteristics corresponding to the PD-L1 antibody and the interpretation methods of different PD-L1 antibodies in different tumors, screen out PDTX tumor biopsy samples that meet the required characteristics from the sample sequencing information stored in the PDTX tumor biopsy database;

[0010] S2. Transfer the CD 34+ HSC cell solution into C-NKG mice to construct a C-NKG mouse humanized immune system HIS model, and inoculate the PDTX tumor biopsy obtained in step S1 into the C-NKG mouse humanized immune system HIS model for passage and amplification to obtain HIS-PDTX tumor biopsy with stable PD-L1 expression;

[0011] Alternatively, inoculate the PDTX tumor biopsy sample obtained in step S1 into C-NKG mice, and transfer peripheral blood mononuclear cells into the mice to obtain hPBMC-PDTX tumor biopsy with stable PD-L1 expression;

[0012] S3. Use the HIS-PDTX tumor biopsy or hPBMC-PDTX tumor biopsy obtained in step 2 as an alternative control area sample for fixation and wax block preparation, staining detection, screen out tumor biopsies with tumor cells ≥ 30% and necrosis ratio ≤ 20%, and interpret the PD-L1 expression level of the HIS-PDTX tumor biopsy or hPBMC-PDTX tumor biopsy;

[0013] S4. Prepare wax blocks and sections from the HIS-PDTX tumor biopsy or hPBMC-PDTX tumor biopsy screened in step S3, and attach the sections to the control area of an adhesive glass slide with a printed label to obtain a tumor tissue PD-L1 gradient quality control slide;

[0014] The tumor tissue PD-L1 gradient quality control slide includes a slide label area, a staining control area, and a tissue attachment area. The slide label area contains the name and product model of the target antibody PD-L1. The staining control area is fixed with three HIS-PDTX tumor living tissues or hPBMC-PDTX tumor living tissue samples with PD-L1 staining gradients for control, namely, PD-L1 staining gradient for negative, PD-L1 staining gradient for medium expression, and PD-L1 staining gradient for high expression.

[0015] Among them, the mouse is a triple severely immunodeficient mouse with T, B, and NK cells, sourced from Cyagen Biosciences Inc., strain name: NOD.Cg-Prkdc scid Il2rg em1cya / Cya, abbreviated as C-NKG.

[0016] When immunohistochemical staining is required, the tissue section of the sample to be tested is adhered to the tissue attachment area and stained synchronously with the control tissue.

[0017] In the technical solution of this application, the HIS-PDTX technology or hPBMC-PDTX technology is used to construct a model, and HIS-PDTX tumor living tissue or hPBMC-PDTX tumor living tissue containing tumor cells and immune cells can be obtained as a PD-L1 control sample. When detecting the expression of PD-L1 by immunohistochemistry, the PD-L1 staining conditions of tumor cells and immune cells can be observed simultaneously. When detecting the expression of PD-L1 in a multi-cell type sample by immunohistochemistry, multiple PD-L1 interpretation methods can be used. Therefore, different antibodies can be selected for PD-L1 staining according to different needs, improving the flexibility and reliability of the tumor tissue PD-L1 gradient quality control slide and providing more reliable data support for immunotherapy research.

[0018] The tissues in the staining control area are sourced from HIS-PDTX tumor living tissues or hPBMC-PDTX tumor living tissues with stable PD-L1 expression stored in the tumor living tissue biobank, and can be revived and amplified using HIS-PDTX or hPBMC-PDTX technology, enabling the acquisition of sufficient tissues for control. Moreover, the tissue source is stable and controllable. Since these living tissue samples stably express PD-L1, as the staining control area, it can ensure its quality and reliability.

[0019] The HIS-PDTX tumor living tissue or hPBMC-PDTX tumor living tissue samples of this application are screened through the RNA-seq information of the samples stored in the tumor living tissue database, and the control standard is more accurate.

[0020] The control sample of the tumor tissue PD-L1 gradient quality control slide in this application has a similar tissue structure to the tissue to be tested, can accurately reflect the distribution of PD-L1, and has higher specificity and sensitivity at the same time; the control tissue of the tumor tissue PD-L1 gradient quality control slide can effectively avoid false positive and false negative results of PD-L1 expression in clinical tests, and can also eliminate the batch effects brought by antibodies of different production batches.

[0021] As a preferred implementation mode of the preparation method of the tumor tissue PD-L1 gradient quality control slide described in this application, in the step S1, the TPM normalization value of the transcriptome sequencing corresponding to the PDTX tumor living tissue sample with a negative PD-L1 staining gradient < 5; the TPM normalization value of the transcriptome sequencing corresponding to the PDTX tumor living tissue sample with a medium expression of PD-L1 is 5-50; the TPM normalization value of the transcriptome sequencing corresponding to the PDTX tumor living tissue sample with a high expression of PD-L1 > 50.

[0022] The HIS-PDTX tumor living tissue or hPBMC-PDTX tumor living tissue sample of this application is screened by using the TPM normalization value of the transcriptome sequencing RNA-seq of the samples stored in the tumor living tissue database, which further improves the accuracy of the PD-L1 control tissue and ensures that the selected samples can more accurately represent the expression of PD-L1.

[0023] As a preferred implementation mode of the preparation method of the tumor tissue PD-L1 gradient quality control slide described in this application, in the step S1, the PD-L1 antibody includes but is not limited to at least one of 22C3 antibody, SP142 antibody, SP263 antibody, WD160 antibody, E1L3N antibody, OR-5E3 antibody, MXR-003 antibody; however, the PD-L1 antibody is not limited to the above examples, and also includes common PD-L1 antibody types in the art, all within the scope of this application;

[0024] The tumor includes but is not limited to at least one of non-small cell lung cancer, urothelial carcinoma, cervical cancer, triple-negative breast cancer; the tumor is not limited to the above tumor types, and also includes common tumor categories in the art, all within the scope of this application;

[0025] The interpretation method includes TPS interpretation method, CPS interpretation method, IC interpretation method or TC+IC interpretation method.

[0026] As a preferred implementation mode of the preparation method of the tumor tissue PD-L1 gradient quality control slide described in this application, the 22C3 antibody uses the TPS interpretation method in non-small cell lung cancer, and the 22C3 antibody uses the CPS interpretation method in urothelial carcinoma or cervical cancer;

[0027] The SP142 antibody uses the IC interpretation method in triple-negative breast cancer and urothelial carcinoma;

[0028] The SP142 antibody uses the TC+IC interpretation method in non-small cell lung cancer;

[0029] The SP263 antibody uses the TC+IC interpretation method in urothelial carcinoma.

[0030] The samples of this application have multiple cell types. Therefore, when performing immunohistochemical staining for PD-L1 expression, multiple methods can be used for interpretation, and different PD-L1 antibodies can be selected for staining according to needs. This diverse PD-L1 detection method helps to improve the accuracy and reliability of PD-L1 detection, thereby more precisely evaluating the expression of PD-L1. Multidimensional observation and flexible selection of PD-L1 antibodies are of great significance for more accurately evaluating the expression of PD-L1 and providing more reliable data support for immunotherapy research.

[0031] As a preferred embodiment of the method for preparing the PD-L1 gradient quality control slide of the tumor tissue described in this application, in step S2, the CD34 + concentration of the HSC cell solution is 5×10 5 cells / ml, and the injection volume is 5×10 4 cells;

[0032] The concentration of the peripheral blood mononuclear cells is 5×10 6 cells / ml, and the injection volume is 5×10 5 cells.

[0033] As a preferred embodiment of the method for preparing the PD-L1 gradient quality control slide of the tumor tissue described in this application, step S2 is specifically:

[0034] Take C-NKG mice within 3 weeks and irradiate them with X-rays at a condition of 100 cGy / min for 2.4 min to clear the marrow. Transfer CD34 5 cells / ml with a concentration of 5×10 + HSC cells into the mice within 4 - 24 h after marrow clearance; start detecting the immune cells in the peripheral blood of the mice every two weeks from the fourth week after supplementing CD34 + HSC cells by flow cytometry. When hCD45 + ≥25% in the peripheral blood of C-NKG mice, it is determined that the construction of the humanized immune system HIS model of C-NKG mice is successful;

[0035] Shear the PDTX tumor living tissue sample obtained in step S1 into 2×2×2 mm 3, inoculate into the HIS model of the humanized immune system of C-NKG mice, and wait until the cumulative volume of the transplanted tumor grows to 800 - 1000 mm 3 , and obtain the HIS-PDTX tumor tissue with stable PD-L1 expression as the alternative control area sample;

[0036] Alternatively, take C-NKG mice at 5 - 8 weeks old and inoculate them with PDTX tumor tissue samples cut into 2×2×2 mm 3 , and wait until the cumulative volume of the transplanted tumor grows to 500 - 800 mm 3 , transfer 100 μl of peripheral blood mononuclear cells with a concentration of 5×10 6 cells / ml into the tumor-bearing C-NKG mice to obtain the hPBMC-PDTX tumor tissue with stable PD-L1 expression as the alternative control area sample.

[0037] By using the HIS-PDTX tumor tissue or the hPBMC-PDTX tumor tissue as the control sample for the PD-L1 gradient quality control slide of the tumor tissue, both human tumor cells and immune cells can be covered, so that the PD-L1 staining conditions of tumor cells and immune cells can be observed simultaneously during immunohistochemical detection of PD-L1. The control sample of the PD-L1 gradient quality control slide of the tumor tissue is similar to the true tissue structure to be detected, can truly reflect the distribution of PD-L1, and has higher specificity and sensitivity.

[0038] As a preferred embodiment of the method for preparing the PD-L1 gradient quality control slide of the tumor tissue described in the present application, in step S3, the PDTX tumor tissue obtained in step S1 is cut into 2×2×2 mm 3 , inoculate it into the HIS model of the humanized immune system of C-NKG mice obtained in step S2 for passage and amplification, and wait until the cumulative volume of the transplanted tumor grows to 800 - 1000 mm 3 , and obtain the HIS-PDTX tumor tissue with stable PD-L1 expression, or cut the PDTX tumor tissue obtained in step S1 into 2×2×2 mm 3 , inoculate it into C-NKG mice, transfer peripheral blood mononuclear cells into the tumor-bearing C-NKG mice, and obtain the hPBMC-PDTX tumor tissue with stable PD-L1 expression.

[0039] The tissues in the staining control region of this application are derived from HIS-PDTX tumor tissues or hPBMC-PDTX tumor tissue samples with stable PD-L1 expression stored in a tumor biopsy sample bank. Using HIS-PDTX or hPBMC-PDTX technology, these samples can be successfully resuscitated and amplified to obtain sufficient tissues for use as controls. This method can ensure the stability and controllability of tissue sources and provide reliable tissue samples for the staining control region. By using these samples as controls, we can more accurately evaluate the expression of PD-L1 and provide reliable results and data support for research.

[0040] As a preferred embodiment of the method for preparing the PD-L1 gradient quality control slide of tumor tissues described in this application, in step S3, the staining includes HE staining or immunohistochemical staining (IHC).

[0041] This application provides a PD-L1 gradient quality control slide of tumor tissues prepared by the method for preparing the PD-L1 gradient quality control slide of tumor tissues described above. The PD-L1 gradient quality control slide of tumor tissues contains lymphocytes with positive PD-L1 expression.

[0042] The PD-L1 gradient quality control slide of tumor tissues designed in this application has control samples with consistent biological characteristics and stability, while ensuring the traceability and continuous supply of sample sources; this application selects real HIS-PDTX tumor tissues or hPBMC-PDTX tumor tissue samples as control samples for the PD-L1 gradient quality control slide of tumor tissues; the tumor tissues prepared in this application contain lymphocytes with positive PD-L1 expression. These samples have real immune microenvironments and heterogeneity characteristics and can accurately reflect the distribution of PD-L1 in HIS-PDTX tumor tissues or hPBMC-PDTX tumor tissue structures. By using these samples as controls, the accuracy and reliability of the PD-L1 gradient quality control slide of tumor tissues can be improved;

[0043] The control samples selected for the PD-L1 gradient quality control slide of tumor tissues are human-derived HIS-PDTX tumor tissues or hPBMC-PDTX tumor tissue samples with a human immune microenvironment. When using immunohistochemical technology to detect PD-L1, the PD-L1 expression of tumor cells and immune cells in tumor tissues can be detected. Therefore, various PD-L1 immunohistochemical interpretation methods can be selected according to needs, and this flexibility reduces the selection limitations for PD-L1 antibodies; clinically, there will be problems of false positives and false negatives in PD-L1 immunohistochemical staining. The PD-L1 gradient quality control slide of tumor tissues provided in this application has stable PD-L1 expression and can avoid the problems of immunohistochemical false positives and false negatives. At the same time, it avoids batch effects caused by different antibodies.

[0044] The present application also provides the use of the above-mentioned tumor tissue PD-L1 gradient quality control slide in detecting PD-L1 in clinical samples, and the clinical sample is a non-living tissue sample.

[0045] Compared with the prior art, the present application has the following beneficial effects:

[0046] The present application provides a tumor tissue PD-L1 gradient quality control slide, its preparation method and application. The tumor tissue PD-L1 gradient quality control slide of the present application provides a control sample with consistent and stable biological characteristics, while ensuring the traceability and continuous supply of the sample source; the present application selects a real HIS-PDTX tumor living tissue or hPBMC-PDTX tumor living tissue sample as the control sample of the tumor tissue PD-L1 gradient quality control slide; the tumor living tissue prepared in the present application contains lymphocytes with positive PD-L1 expression. These samples have real immune microenvironment and heterogeneity characteristics, and can accurately reflect the distribution of PD-L1 in the HIS-PDTX tumor living tissue or hPBMC-PDTX tumor living tissue structure. By using these samples as controls, the accuracy and reliability of the tumor tissue PD-L1 gradient quality control slide can be improved; when detecting PD-L1 using immunohistochemistry technology, the PD-L1 expression of tumor cells and immune cells in the tumor tissue can be detected. Therefore, a variety of PD-L1 immunohistochemistry interpretation methods can be selected according to needs, and this flexibility reduces the selection limitation for PD-L1 antibodies. The PD-L1 detection control product provided by the present application has stable PD-L1 expression, can avoid the problems of immunohistochemical false positives and false negatives. At the same time, it avoids batch effects caused by different antibodies. Description of the Drawings

[0047] Figure 1 It is a flow chart of the resuscitation, amplification and sampling of the HIS-PDTX tumor living tissue modeling in step 2 of Example 1;

[0048] Figure 2 It is a flow chart of the resuscitation, amplification and sampling of the hPBMC-PDTX tumor living tissue modeling in step 2 of Example 1;

[0049] Figure 3 It is a HE staining diagram of the tumor tissue and an immunohistochemical staining diagram of PD-L1 in the tumor tissue;

[0050] Figure 4 It is a schematic diagram of the tumor tissue PD-L1 gradient quality control slide;

[0051] Figure 5 It is an immunohistochemical staining diagram of the tumor tissue PD-L1 gradient quality control slide prepared in Example 2. Detailed Embodiments

[0052] To better illustrate the purpose, technical solution, and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0053] In the following embodiments, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0054] Example 1. Screening of HIS-PDTX tumor biopsies or hPBMC-PDTX tumor biopsies with stable PD-L1 expression

[0055] Step 1: Screening of tumor tissues:

[0056] So far, the scoring of immune cells has been limited to the clone SP142 in the treatment of triple-negative breast cancer and urothelial cancer with atezolizumab. The score is defined as the proportion of the tumor area occupied by PD-L1-stained immune cells of any intensity (intratumoral and adjacent peritumoral stroma, excluding necrosis). The CPS score is defined as the comprehensive positive score calculated by measuring the PD-L1 staining of tumor cells and tumor-associated immune cells (lymphocytes, histiocytes), dividing by the total number of tumor cells, and then multiplying by 100. This interpretation method can be used with the 22C3 antibody in cervical cancer and urothelial cancer. In addition, the TPS interpretation method is used in non-small cell lung cancer, that is, the PD-L1 positive staining of tumor cells. The immune cell (IC) interpretation method can be used with the SP142 antibody in triple-negative breast cancer and urothelial cancer; the combined tumor cell (TC) and immune cell (IC) scoring using SP142 staining is used for the treatment of non-small cell lung cancer with atezolizumab, or SP263 staining is used for the treatment of urothelial cancer with durvalumab.

[0057] Table 1 Antibodies and PD-L1 interpretation methods in different tumors

[0058]

[0059] According to the transcriptome sequencing (RNA-seq) information characteristics corresponding to PD-L1, biopsy samples meeting the following characteristic requirements are screened through the sample sequencing information stored in the PDTX tumor biopsy database self-built by Puenrui, as shown in Table 2.

[0060] Table 2 RNA-seq information characteristics

[0061]

[0062] Step 2: Resuscitation, amplification, and sampling of HIS-PDTX tumor biopsies

[0063] (1) Fresh umbilical cord blood is delivered to the laboratory within 24 hours after blood collection. Then, the blood is diluted 1:3 with phosphate buffered saline (PBS), and mononuclear cells are separated by density gradient centrifugation on Ficoll medium.

[0064] (2) The mononuclear cells are separated by CD34 + microbead kit to obtain CD34 + HSC cell solution. The CD34 + HSC cell solution is adjusted to a concentration of 5×10 5 cells / ml, and then the 5×10 5 cells / ml CD34 + HSC cell solution is stored at 2 - 8°C until injection.

[0065] (3) Take C-NKG mice within 3 weeks (100 cGy / min × 2.4 min, irradiated with X-rays for myeloablation), and inject 5×10 5 cells / ml CD34 + HSC cells into the mice through the tail vein of the mice within 4 - 24 hours after myeloablation. The injection volume is 100 μl (5×10 4 cells).

[0066] (4) Start detecting immune cells in the peripheral blood of mice by flow cytometry 4 weeks after supplementing CD34 + HSC cells. The time interval is two weeks. When hCD45 + ≥ 25% in the peripheral blood of C-NKG mice, it is determined that the construction of the humanized immune system HIS model of C-NKG mice is successful.

[0067] (5) While constructing the humanized immune system HIS model of C-NKG mice, cut the PDTX tumor living tissue that meets the requirements after screening into 2×2×2 mm 3 , and inoculate it subcutaneously on the back of C-NKG mice. Inoculate the tumor tissue sample that has been passed to the P5 passage and has a stable growth rate subcutaneously into the humanized immune system HIS model of C-NKG mice.

[0068] (6) After inoculation, observe the animal status every day, measure the tumor volume once a week and weigh the tumor-bearing mice, and calculate the tumor volume V (mm 3 ) = (a × b 2 ) / 2, where a: the long diameter of the tumor, b: the short diameter of the tumor.

[0069] (7) Wait until the cumulative volume of the transplanted tumor grows to 80 mm 3, anatomically excise and cut, and randomly inoculate (P1 generation) subcutaneously into the humanized immune system HIS model of C-NKG mice for subculture and amplification.

[0070] (8) Wait until the cumulative volume of the transplanted tumor grows to 800 - 1000 mm 3 , anatomically excise and use as a sample for the alternative control area for paraffin embedding and section preparation. The flow chart is as Figure 1 shown.

[0071] Or,

[0072] Step 2: Resuscitation, amplification, and sampling of hPBMC-PDTX tumor biopsy modeling:

[0073] (1) Cut the selected PDTX tumor biopsy that meets the requirements into 2×2×2 mm 3 , and inoculate it subcutaneously on the back of C-NKG mice.

[0074] (2) The freshly collected peripheral blood should be delivered to the laboratory within 24 hours after blood collection, diluted (peripheral blood: phosphate buffer or RPMI1640 = 1:2), and peripheral blood mononuclear cells are separated by density gradient centrifugation on Ficoll medium.

[0075] (3) Count the above-mentioned peripheral blood mononuclear cells, and then adjust the concentration of peripheral blood mononuclear cells to 10×10 6 / ml.

[0076] (4) Wait until the cumulative volume of the tumor in C-NKG mice grows to 500 - 800 mm 3 After that, inject peripheral blood mononuclear cells through the tail vein, and the injection volume is 100 μl (5×10 5 cells).

[0077] (5) Anatomically excise and use as a sample for the alternative control area for paraffin embedding and section preparation. The flow chart is as Figure 2 shown.

[0078] Step 3: Fixation and wax block preparation of PDTX tumor biopsy samples:

[0079] (1) Tissue fixation: Add the HIS-PDTX tumor biopsy or hPBMC-PDTX tumor biopsy to 10 times the volume of 10% neutral formaldehyde solution and fix at room temperature for 12 - 24 h.

[0080] (2) Tissue sampling: Cut the fixed tissue into tissue blocks of ≤1.5 cm×1.5 cm×0.5 cm, put them into the embedding cassette with printed labels, and put them into the tissue dehydration basket.

[0081] (3) Tissue dehydration: The tissue was dehydrated by immersing it successively in ethanol with different concentration gradients: 70% ethanol for 1 h → 80% ethanol for 2 h → 85% ethanol for 70 min → 95% ethanol for 1 h → 95% ethanol for 50 min → absolute ethanol for 1 h → absolute ethanol for 50 min.

[0082] (4) Tissue clearing: The tissue after gradient ethanol dehydration was successively immersed in xylene for clearing: xylene I for 30 min → xylene II for 25 min.

[0083] (5) Tissue infiltration with paraffin: The tissue after tissue clearing was immersed in the melted paraffin for tissue infiltration with paraffin: paraffin I for 1 h → paraffin II for 1 h → paraffin III for 1 h.

[0084] (6) Tissue embedding: Tissue embedding was carried out in a tissue embedding machine. A little melted paraffin was dropped into a preheated stainless - steel embedding mold. After the paraffin at the bottom had slightly solidified, the tissue was placed at the bottom of the mold. The position of the tissue was adjusted, and the tissue was gently pressed to make it on the same plane. The embedding cassette was placed over the mold, and the wax liquid was continuously filled. It was transferred to a pre - cooled freezing table and cooled for 30 min. The wax block was removed, and the excess paraffin was trimmed.

[0085] Step 4: HE staining:

[0086] (1) The wax block was placed in the slot of the microtome for sectioning. 4 - μm white sections were cut and transferred to a water bath at about 45 °C until the sections were completely flattened.

[0087] (2) After flattening, the tissue was adhered to the glass slide.

[0088] (3) After slightly drying, it could be baked on a baking machine, generally baked on a baking machine at about 60 °C for 15 - 30 min, and the baking time could be extended for special samples.

[0089] (4) After baking, it was placed in a Leica ST5010 automatic stainer for staining.

[0090] (5) The reagent sequence and staining time for staining with the Leica ST5010 automatic stainer are shown in Table 3 below:

[0091] Table 3

[0092]

[0093] (6) The stained sections were transferred to a Leica CV5030 cover - slip mounting machine for cover - slipping.

[0094] (7) Microscopic observation: Samples with a tumor cell ratio < 30% and a necrosis ratio > 20% were excluded, referring to Figure 3It is an example of the HE staining result of tumor tissue. Figure a is a schematic diagram of an unqualified tumor sample, with the tumor cell content < 30% and the necrosis ratio > 20%; Figure b is a schematic diagram of a qualified tumor sample, with the tumor cell content ≥ 30% and the necrosis ratio ≤ 20%.

[0095] Step 5: Immunohistochemical staining (IHC):

[0096] (1) Tissue sectioning: After placing the obtained wax block on the cold table for 20 minutes, place it on the microtome for sectioning. First, trim the wax block: In the trimming mode, the trimming section thickness can be adjusted to 15 - 20 μm. After trimming the wax block until the tissue is fully exposed, adjust the section thickness to about 4 μm, and then perform sectioning. The sections must be ensured to be complete, uniform, without knife marks, wrinkles, and cracks.

[0097] (2) Spreading the sections: Gently pick up the sections with a brush and transfer them to the water surface in the spreading machine with forceps to spread (temperature 45°C. During the process of transferring the sections to the water surface of the spreading machine, the sections can rotate but not flip).

[0098] (3) Picking up the sections: Attach the spread sections to the glass slides. When picking up the sections, hold the frosted area of the glass slide with the right hand to avoid generating air bubbles between the sections and the glass slides as much as possible. Tilt the glass slide to 45° to let the excess water on the sections flow down.

[0099] (4) Baking the sections: Place the glass slides on the baking machine and bake at 60°C for 60 minutes to make tissue paraffin sections.

[0100] (5) Dewaxing: After the baking is completed, quickly transfer the paraffin sections to xylene. Xylene I for 5 minutes → Xylene II for 5 minutes → Xylene III for 5 minutes.

[0101] (6) Hydration: Take out the sections from xylene, drain them, and transfer them to ethanol with different concentration gradients for hydration. Absolute ethanol I for 5 minutes → Absolute ethanol II for 5 minutes → 95% ethanol for 5 minutes → 75% ethanol for 5 minutes, and then wash with water after completion.

[0102] (7) Antigen retrieval: Add an appropriate amount of antigen retrieval solution (Shenzhen Dameng, Guangdong Medical Device Preparation 20221796) to the staining cylinder for heat retrieval. Boil for 10 minutes, and after natural cooling, rinse with PBS buffer 3 times, 3 minutes each time.

[0103] (8) Blocking: Shake off the excess PBS buffer from the sections, add the endogenous peroxidase blocking reagent, and drip 100 μl onto the tissue. Incubate at 37°C for 10 minutes, and rinse with PBS buffer 3 times, 3 minutes each time.

[0104] (9)Primary antibody incubation: Drain the excess PBS buffer from the sections, add 100 μl of diluted PD-L1 antibody, incubate at 37 °C for 1 h, and rinse with PBS buffer 3 times, 3 min each time.

[0105] (10)Secondary antibody incubation: Drain the excess PBS buffer from the sections, add 100 μl of secondary antibody, incubate at room temperature for 20 min, and rinse with PBS buffer 3 times, 3 min each time.

[0106] (11)BAB color development: Drain the excess PBS buffer from the sections, add 100 μl of DAB mixture (Solution A: Solution B = 1:1), incubate for 5 - 8 min, and stop the color development by washing with water.

[0107] (12)Nuclear counterstaining: Drain the excess liquid from the sections, add 100 μl of hematoxylin stain, stain for 20 - 60 s (the staining time can be adjusted according to the actual situation), and rinse with running water for 5 min.

[0108] (13)Differentiation (optional depending on the nature of hematoxylin): Immerse the sections in the differentiation solution for 2 - 5 s, quickly take out the sections, and rinse the slides with tap water for 5 min.

[0109] (14)Dehydration and clearing: Drain the water from the rinsed sections, dehydrate with gradient ethanol in the following order: 75% ethanol: 5 min → 95% ethanol: 5 min → absolute ethanol: 5 min. Take out the sections from absolute ethanol and place them in xylene I for 5 min → xylene II for 5 min → xylene III for 5 min for clearing treatment (this process is carried out in a fume hood).

[0110] (15)Sealing with neutral balsam: Take out the sections and place them in a fume hood. Before the xylene evaporates completely, add neutral balsam dropwise and cover with a coverslip (this process is carried out in a fume hood).

[0111] (16)Observation under microscope: Select tissue samples with PD-L1 results judged as negative, medium expression, and high expression respectively.

[0112] Example 2. A PD-L1 gradient quality control slice for PDTX tumor tissue and its preparation method

[0113] (1)Preparation of wax blocks: Use a tissue microarray instrument to take 1 mm of the above HIS-PDTX tumor living tissue or hPBMC-PDTX tumor living tissue with stable expression of PD-L1 (negative, medium expression, and high expression), and place it in a pre-cast wax block. Place the wax block with the tissue in a stainless steel bottom mold, pay attention to the direction: the cut surface is facing down, place it in an oven at 70 °C for 30 - 60 min, wait until the wax block is completely transparent, and take out the wax block. Place the tissue embedding cassette above the stainless steel mold, continue to pour wax until the bottom of the cassette is completely immersed, and let it cool.

[0114] (2) Tissue sectioning: Place the wax block on a cold stage for 20 minutes, then place it on a microtome for sectioning. Trim the wax block first: In the trimming mode, the thickness of the trimmed slices can be adjusted to 15-20 μm. After trimming the wax block until all three tissues are exposed and on the same plane, adjust the slice thickness to about 4 μm, and then section. The slices must be complete, uniform, without knife marks, wrinkles, or cracks.

[0115] (3) Spreading the slice: Use a brush to gently pick up the slice, and use tweezers to transfer the slice to the water surface in the slice spreader and spread it (temperature 45°C. During this process, it is necessary to ensure that the slice is not flipped).

[0116] (4) Fishing out the slice: Place the fully unfolded slice on an adhesive slide. When fishing out the slice, hold the frosted area of ​​the slide with your right hand and try to avoid bubbles between the slice and the slide. After fishing out the slice, tilt the slide 45 degrees to allow excess water on the slice to flow out.

[0117] This embodiment provides a tumor tissue PD-L1 gradient quality control sheet, which includes a slide label area, a staining control area and a tissue patch area. Figure 4 The slide label area contains the name and product model of the target antibody PD-L1 corresponding to the immunohistochemical slide with control; the staining control area is fixed with 3 kinds of PDTX tumor living tissue samples with different PD-L1 staining gradients of negative, medium expression and high expression as controls; the staining control area is divided into 3 independent blocks from top to bottom, which are the staining control areas with negative PD-L1 staining gradient, medium expression PD-L1 staining gradient and high expression PD-L1 staining gradient; when immunohistochemical staining is required, the tissue section of the sample to be tested is adhered to the tissue patch area.

[0118] In some specific embodiments, the control tissue is stained simultaneously with the test tissue during the immunohistochemical staining process. Figure 5 The immunohistochemical staining images of the PD-L1 gradient quality control film of tumor tissue. a, b, and c are the immunohistochemical staining images of tumor tissue sections with negative, medium expression, and high expression of PD-L1, respectively.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A method for preparing a tumor tissue PD-L1 gradient quality control sheet, characterized in that: The following steps are involved: S1. Based on the RNA-seq information characteristics of the transcriptome sequencing corresponding to the PD-L1 antibody and the interpretation methods of different PD-L1 antibodies in different tumors, the PDTX tumor biopsy samples that meet the characteristic requirements are screened out through the sample sequencing information stored in the PDTX tumor biopsy database; S2. Put the CD 34+ HSC cell fluid was transferred into C-NKG mice to construct a C-NKG mouse humanized immune system HIS model, and the PDTX tumor living tissue obtained in step S1 was inoculated into the C-NKG mouse humanized immune system HIS model for passage and amplification to obtain HIS-PDTX tumor living tissue with stable expression of PD-L1; Alternatively, the PDTX tumor biopsy sample obtained in step S1 is inoculated into C-NKG mice, and peripheral blood mononuclear cells are transferred into the mice to obtain hPBMC-PDTX tumor biopsy with stable expression of PD-L1; S3, fix the HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue obtained in step S2 as a sample of the alternative control area, prepare a wax block, stain and detect, screen out tumor biopsy tissues with tumor cells ≥30% and necrosis ratio ≤20%, and interpret the PD-L1 expression level of the HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue; S4, preparing wax blocks and slices of the HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue screened in step S3, and attaching the slices to the control area on the adhesive slide with a printed label to obtain a tumor tissue PD-L1 gradient quality control film; The tumor tissue PD-L1 gradient quality control sheet includes a slide label area, a staining control area and a tissue patch area, wherein the slide label area contains the name and product model of the target antibody PD-L1; the staining control area is fixed with three HIS-PDTX tumor biopsy tissue samples or hPBMC-PDTX tumor biopsy tissue samples, which serve as controls and have a negative PD-L1 staining gradient, a medium expression PD-L1 staining gradient and a high expression PD-L1 staining gradient; In step S1, the TPM normalized value of the transcriptome sequencing corresponding to the PDTX tumor biopsy sample with a negative PD-L1 staining gradient is <5; the TPM normalized value of the transcriptome sequencing corresponding to the PDTX tumor biopsy sample with a medium expression PD-L1 staining gradient is 5-50; the TPM normalized value of the transcriptome sequencing corresponding to the PDTX tumor biopsy sample with a high expression PD-L1 staining gradient is >50; The determination method includes a TPS determination method, a CPS determination method, an IC determination method or a TC+IC determination method.

2. The method for preparing the tumor tissue PD-L1 gradient quality control sheet according to claim 1, characterized in that: In step S1, the PD-L1 antibody includes but is not limited to at least one of 22C3 antibody, SP142 antibody, SP263 antibody, WD160 antibody, E1L3N antibody, OR-5E3 antibody, and MXR-003 antibody; The tumor includes but is not limited to at least one of non-small cell lung cancer, urothelial carcinoma, cervical cancer, and triple-negative breast cancer.

3. The method for preparing the tumor tissue PD-L1 gradient quality control sheet according to claim 2, characterized in that: The 22C3 antibody is analyzed by TPS in non-small cell lung cancer, and the 22C3 antibody is analyzed by CPS in urothelial carcinoma or cervical cancer; The SP142 antibody uses an IC interpretation method in triple-negative breast cancer and urothelial carcinoma; The SP142 antibody adopts the TC+IC interpretation method in non-small cell lung cancer; The SP263 antibody adopts the TC+IC interpretation method in urothelial carcinoma.

4. The method for preparing the tumor tissue PD-L1 gradient quality control sheet according to claim 1, characterized in that: In step S2, the CD34 + The concentration of HSC cell solution was 5×10 5 cells / ml, injection volume was 5×10 4 cells; The concentration of peripheral blood mononuclear cells was 5×10 6 cells / ml, injection volume was 5×10 5 cells.

5. The method for preparing the tumor tissue PD-L1 gradient quality control sheet according to claim 1, characterized in that: The step S2 is specifically as follows: C-NKG mice within 3 weeks were irradiated with X-rays at 100 cGy / min for 2.4 min to ablate the myeloablation. Within 4 to 24 hours after ablation, a concentration of 5×10 5 CD34 cells / ml + HSC cells; after supplementation with CD34 + Starting from the fourth week after HSC cells were generated, immune cells in the peripheral blood of mice were detected by flow cytometry every two weeks. + When the ratio is ≥25%, the humanized immune system HIS model of C-NKG mice was successfully constructed. The PDTX tumor biopsy samples obtained in step S1 were cut into 2×2×2 mm 3 , inoculated into the C-NKG mouse humanized immune system HIS model, and waited for the transplanted tumor to grow to a cumulative volume of 800-1000mm 3 , obtain HIS-PDTX tumor biopsies with stable PD-L1 expression as alternative control area samples; Alternatively, take 5-8 week old C-NKG mice and cut into 2 × 2 × 2 mm 3 PDTX tumor biopsy samples, the transplanted tumor volume accumulates to 500~800mm 3 100 μl of 5×10 6 hPBMC-PDTX tumor biopsies with stable expression of PD-L1 were obtained as alternative control samples.

6. The method for preparing the tumor tissue PD-L1 gradient quality control sheet according to claim 1, characterized in that: In the step S3, the staining includes HE staining or immunohistochemistry staining (IHC).

7. The tumor tissue PD-L1 gradient quality control sheet prepared by the method for preparing the tumor tissue PD-L1 gradient quality control sheet according to any one of claims 1 to 6, characterized in that: The tumor tissue PD-L1 gradient quality control film contains lymphocytes that are positive for PD-L1 expression.

8. Use of the tumor tissue PD-L1 gradient quality control sheet according to claim 7 in the preparation of a clinical sample product for detecting PD-L1, characterized in that: The clinical sample is a non-living tissue sample.

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