A detection method for a solid tumor targeting index and a reagent combination thereof

Abstract

The application provides a solid tumor targeting index detection method and a reagent combination thereof, including detection of a solid tumor targeting therapy molecular marker and detection of a solid tumor microenvironment, and the evaluation method comprises the following steps: collecting a fresh tumor tissue sample; preparing a tumor tissue sample to be detected into a single cell suspension; establishing a flow cytometry on-machine model; detecting the solid tumor targeting therapy molecular marker by using flow cytometry; and detecting the solid tumor microenvironment by using flow cytometry. The application has the advantages of less required samples, short detection time, and the ability to provide information about various factors that are helpful for prognosis and treatment, including quantification of tumor infiltrating lymphocytes, the proportion of tumor cells with positive expression of PD-L1, EGFR and ALK, and the proportion of tumor cells with co-expression.

Description

Technical Field

[0001] This invention relates to the detection of tumor cell markers and driver genes in solid lung cancer, specifically providing a method for detecting lung cancer tumor-targeting indicators and a reagent combination for detecting tumor and its microenvironment markers in the method. Background Technology

[0002] Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for the vast majority (80%-85%). Although increased health awareness and the application of early screening technologies such as spiral CT have reduced the proportion of newly diagnosed lung cancer patients with intermediate or advanced-stage tumors, the heterogeneity of tumors and poor treatment outcomes in advanced stages result in a poor prognosis, with a 5-year relative survival rate of only 16.1%. How to more effectively prolong the survival rate and reduce mortality in lung cancer patients remains a key focus in lung cancer treatment. Different histological subtypes and different driver genes in patients with the same stage of NSCLC can lead to different treatment effects and prognoses with the same treatment approach. For most NSCLC patients, surgical resection remains the preferred treatment. However, in patients with advanced NSCLC, the lesions have usually metastasized, making surgery ineffective. Accurate staging, subtype determination, analysis of molecular lung cancer markers, and detection of driver genes in advanced NSCLC, along with the selection of appropriate treatment methods and targeted drugs, can effectively improve the prognosis and survival rate of NSCLC patients.

[0003] With platinum-based chemotherapy for NSCLC reaching a plateau, macromolecular targeted therapies, such as epidermal growth factor tyrosine kinase inhibitors, have significantly prolonged progression-free survival in driver gene-positive advanced NSCLC. However, the instability of driver genes associated with NSCLC development and progression means that targeted drugs are unlikely to cure the tumor. Immunotherapy, as a new hope for cancer treatment, has significantly improved the 5-year survival rate of advanced NSCLC, although some patients experience poor prognosis.

[0004] Activation of T cells in the immune system requires at least two signals: the first is the recognition by the T cell antigen receptor (TCR) of a specific peptide presented by antigen-presenting cells (APCs) that binds to the major histocompatibility complex (MHC); the second is the linkage of a co-stimulatory molecule (such as CD28) with its selective ligand B7 on the APC. Both signals are indispensable and work together to promote T cell activation, cytokine secretion, and immune function. T cell activation is not unlimited. After a certain period of activation, the inhibitory co-stimulatory molecule PD-1 expressed on activated T cells inhibits T cell activation and mediates T cell apoptosis. These positive and negative co-stimulatory molecule signals determine the intensity and flexibility of the immune response. Immunotherapy for tumors refers to stimulating or mobilizing the body's immune system to enhance the anti-tumor immunity of the tumor microenvironment, thereby controlling and killing tumor cells.

[0005] Epidermal growth factor receptor (EGFR) is a receptor-type tyrosine kinase expressed on the cell membrane. Under normal conditions, the extracellular terminus of EGFR binds to its ligand to form a dimer, while the intracellular terminus binds to ATP and undergoes phosphorylation, activating various downstream signaling pathways, such as PKC, JAK, and ERK, to regulate various physiological processes including cell growth, differentiation, and development. EGFR overexpression and mutations can lead to activation of the EGFR domain, abnormally activating downstream signaling pathways and promoting malignant proliferation of tumor cells. EGFR is considered one of the effective targets for cancer treatment, and many anti-EGFR inhibitors have been developed and researched, including EGFR monoclonal antibodies such as cetuximab, nimotuzumab, and panitumumab, and small molecule tyrosine kinase inhibitors (TKIs) such as erlotinib, gefitinib, and icotinib.

[0006] The anaplastic lymphoma kinase (ALK) gene is considered a potent cancer driver gene because ALK gene rearrangements have been detected in hematologic malignancies and solid tumors, including anaplastic large cell lymphoma, fibromyalgia, and non-small cell lung cancer (NSCLC), indicating its involvement in tumorigenesis and development. The ALK gene is primarily expressed during the embryonic period, promoting nerve cell proliferation and playing a role in the development of the brain and peripheral nervous system. Once the nervous system is fully developed, it typically enters a dormant state, and other cells in the body generally do not express ALK. ALK activation usually occurs through chromosomal rearrangements.

[0007] Tumors reside within the tumor microenvironment, which includes the tumor itself, immune cells, and stroma; these elements interact to determine the fate of specific cancers. Therefore, cancer treatment requires precision medicine to collect and evaluate all tumor-related information. Currently, methods for detecting important molecular targets for lung cancer treatment, such as PD-L1, EGFR, and ALK, include immunohistochemistry, sequencing, PCR, and FISH. These detection technologies have drawbacks such as numerous steps required, large sample volumes, and high costs. Traditional methods are single-detection methods, providing only relatively limited information and making it difficult to detect multiple tumor biomarkers.

[0008] Flow cytometry involves preparing a single-cell suspension of the sample, staining it with a specific fluorescent dye, and then placing it in a sample tube for flow cytometry. The stained cells are discharged as single cells as they pass through the laser focusing area and are excited by the incident laser, producing fluorescence at a specific wavelength. By analyzing the fluorescence, light scattering, light absorption, or cell resistance signals collected by the instrument, the state, type, and DNA content of the cells can be distinguished. Flow cytometry combined with monoclonal antibodies can quantitatively detect cell surface and intracellular antigens, oncogene proteins, and membrane receptors. Clinically, flow cytometry is used to detect the body's immune status, residual microfocal lesions, and to assess the effectiveness of drug treatment and prognosis. Tumors are clinically classified as solid tumors and non-solid tumors. Solid tumors are tangible masses that can be detected through clinical examinations such as X-rays, CT scans, ultrasound, or palpation. Non-solid tumors are tumors that cannot be seen or palpated by X-rays, CT scans, ultrasound, or palpation; leukemia, a hematological disease, is an example of a non-solid tumor. In current technologies, flow cytometry is primarily used for blood samples and in vitro cultured cell samples, and is rarely used for tissue type analysis. For solid tumor samples, flow cytometry typically involves physical grinding or enzymatic digestion to obtain single-cell suspensions. However, these methods damage the cell surface structure, disrupting the composition and quantity of surface proteins. This results in low single-cell suspension yields and affects the accuracy and sensitivity of the detection. Therefore, flow cytometry is difficult to use for clinical detection of solid tumors. Summary of the Invention

[0009] To overcome the above-mentioned shortcomings, the present invention provides an evaluation method for targeted therapy of solid tumors, including the detection of molecular markers for targeted therapy of solid tumors and the detection of immune cells surrounding solid tumors. The evaluation method includes the following steps:

[0010] Step 1: Collect fresh tumor tissue samples;

[0011] Step 2: Prepare a single-cell suspension from the tumor tissue sample to be tested;

[0012] Step 3: Establish a flow cytometry model;

[0013] Step 4: Detect molecular markers for targeted therapy of solid tumors using flow cytometry;

[0014] Step 5: Use flow cytometry to detect immune cells surrounding solid tumors;

[0015] The order of steps four and five is not important.

[0016] In existing technologies, the evaluation of tumor treatment is limited to the detection of tumor cells themselves. The evaluation method provided in this application not only detects biomarkers of tumor cells themselves but also detects immune biomarkers in the tumor cell microenvironment. This allows for a systematic and comprehensive evaluation of tumor drugs and / or treatment regimens. The solid tumor microenvironment described in this application refers to the immune microenvironment inhabited by solid tumor cells. Solid tumors include both tumor cells and non-tumor cells, with the non-tumor cells constituting the tumor cell microenvironment. Furthermore, the solid tumor microenvironment described in this invention involves detecting immune cells surrounding tumor tissue obtained during surgery, aiming to determine the nature of the tumor's microenvironment in the human body. More preferably, the detection of the solid tumor microenvironment in this invention refers to the detection of immune cells surrounding the solid tumor. More preferably, the detection of changes in the percentage of CD4+ and CD8+ cells, and / or changes in PD-1 on related immune cells, is also preferred. The microenvironment described in this invention differs from the traditional understanding of the microenvironment. Instead of detecting changes in immune cells throughout the peripheral blood, this invention focuses on the expression of immune cells at the tumor site, providing a more precise reflection of the solid tumor microenvironment. The evaluation provided in this application can detect changes in tumor cells before and / or after treatment, as well as changes in the tumor cell microenvironment, including but not limited to the types of immune cells in the microenvironment, the types and levels of cytokines expressed by each immune cell. This invention innovatively uses flow cytometry to detect molecular biomarkers for targeted therapy of solid tumors.

[0017] Preferably, in step four, the molecular group of molecular markers for targeted therapy of solid tumors includes: CD45, PD-L1, EGFR, ALK, and Cell Cycle Dye. Among them, EGFR and ALK are the most common driver genes for lung cancer, accounting for the vast majority of non-small cell lung cancer patients in Asia. PD-L1 is an immune screening point used to determine whether a patient is suitable for immunotherapy and prognosis.

[0018] Cell cycle dye (CCY) can detect whether cells are aneuploid or diploid, and together with CD45, it can distinguish between solid tumor cells and non-tumor cells. This invention is the first to apply euploidy detection combined with CD molecular methods to flow cytometry for solid tumor detection, specifically the combined application of CD45 and Cell Cycle Dye to distinguish between tumor cells and non-tumor cells. This differs from flow cytometry methods for detecting hematologic malignancies, which primarily use forward scattering (FSC) and side-scattering (SSC) methods combined with CD molecules for differentiation.

[0019] By detecting the expression levels of the tumor marker PD-L1 and the driver genes EGFR and ALK, this invention helps clinicians assess disease progression, select appropriate medications, and predict prognosis in cancer patients, thus achieving the goal of auxiliary diagnosis. In existing technologies, clinical methods such as PCR, first-generation sequencing, and fluorescence in situ hybridization can detect the presence of pathogenic mutations in EGFR and ALK genes in tumor tissue samples. Immunohistochemistry can measure the expression levels of these three marker proteins in tumor tissue, thereby assessing the patient's disease progression. However, these methods can only obtain the detection results of a single marker per sample, meaning that only one marker can be detected per tissue sample per experiment. In contrast, this invention can simultaneously obtain the detection results of all three markers in a single tissue sample experiment. Compared to existing clinical testing technologies, achieving the same detection effect requires fewer tissue samples, less manpower, and less time. From a methodological perspective, flow cytometry is more accurate than immunohistochemistry, avoiding subjective factors in interpreting positive and negative results, and providing the percentage of tumor cells expressing PD-L1, ALK, and EGFR in the experimental sample. The method provided in this application utilizes flow cytometry to simultaneously detect three biomarkers—PD-L1, EGFR, and ALK—as well as CD45 and Cell Cycle Dye, and the results reflect the expression levels of PD-L1, EGFR, and ALK proteins in tumor samples. This invention employs flow cytometry for detection, requiring only a single tissue sample to simultaneously detect the aforementioned molecular biomarkers in tumors, offering advantages such as high accuracy, short detection time, and the use of a small number of tissue samples.

[0020] CD45 molecules are expressed on all leukocytes and are called leukocyte common antigen (LCA). CD45 is composed of a class of structurally similar, large-molecule transmembrane proteins widely distributed on the surface of leukocytes. Its cytoplasmic segment acts as a protein tyrosine phosphatase, activating the substrates P56lck and P59fyn by dephosphorylation of tyrosine residues, playing a crucial role in cellular signal transduction. CD45 is a key molecule in cell membrane signal transduction, playing a vital role in lymphocyte development, maturation, functional regulation, and signal transduction. The distribution of CD45 can serve as a classification marker for certain T cell subsets. In this invention, CD45 is used to distinguish tumor cells in solid tumors; specifically, CD45-negative cells are identified as tumor cell populations.

[0021] The tumor marker PD-L1 is a ligand that co-inhibits the programmed cell death receptor PD-1, also known as programmed cell death 1 ligand 1, and is expressed on many tumor cells, including non-small cell lung cancer (NSCLC). The abundant expression of PD-L1 on the surface of tumor cells binds to PD-1 on T cells, preventing T cells from recognizing tumor cells, escaping T cell "killing," and avoiding a strong immune response, thus maintaining growth and proliferation. Based on this, tumor immunotherapy works by targeting drugs to competitively bind to PD-L1 on tumor cells or PD-1 on T cells, preventing the binding of PD-L1 on tumor cells to PD-1 on T cells, and ensuring the normal activation of the immune response in cancer patients. The NCCN treatment guidelines have listed PD-L1 as a biomarker for NSCLC, serving as an essential immune screening point for advanced NSCLC patients, and requiring IHC to detect PD-L1 expression levels in tumor tissue of at least 50%. Low PD-L1 expression in tumor tissue is one of the predictive markers of poor immunotherapy efficacy.

[0022] EGFR is a driver gene in non-small cell lung cancer (NSCLC). EGFR overexpression has been shown to be associated with prognosis and metastatic spread in various cancers, with most EGFR overexpression significantly associated with lymph node metastasis and later pathological stages. In lung cancer, EGFR protein overexpression and EGFR gene amplification are more common in adenocarcinoma, with 70% of adenocarcinoma patients exhibiting EGFR overexpression. EGFR gene expression levels vary significantly among NSCLC patients at different stages. Studies in NSCLC have found that EGFR expression is associated with reduced survival. EGFR overexpression in advanced NSCLC patients is associated with cancer cell survival, metastasis, invasion, and chemotherapy resistance, generally indicating poor survival and a poor prognosis.

[0023] ALK is also a driver gene in non-small cell lung cancer (NSCLC), with ALK rearrangements occurring in approximately 1%-7% of NSCLC patients. Currently, various ALK rearrangements have been identified clinically, with the ALK-EML4 rearrangement being the most common in NSCLC. Methods for detecting ALK rearrangements mainly include fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and polymerase chain reaction (PCR). FISH can specifically and sensitively detect ALK rearrangements and is currently the most commonly used method for clinical detection of ALK fusion genes. PCR can sensitively detect known types of ALK rearrangements. The accuracy and sensitivity of IHC in detecting ALK rearrangement expression largely depend on the antibody and is usually used for initial screening of ALK gene rearrangements. The 2018 Chinese guidelines for the treatment of non-small cell lung cancer designated the VENTANA ALK (D5F3) IHC test kit as the first-line routine diagnostic method for ALK-positive NSCLC, distinguishing it from the routine IHC method used for initial screening. However, after routine IHC initial screening, either FISH or VENTANA ALK or PCR should be performed for confirmation.

[0024] Preferably, in step five, the molecular combination of solid tumor microenvironment detected includes CD3, CD4, CD8, CD45, and PD-1. Currently, conventional detection techniques for immunotherapy targets are immunohistochemical staining or PCR. CD3, CD4, CD8, and CD45 are relevant indicators for immunological detection in hematologic malignancies (i.e., non-solid tumors). The advantage of this invention lies in its innovative use of flow cytometry to detect the immune environment surrounding solid tumors and the expression of immune cells, especially through the detection of markers CD3, CD4, CD8, CD45, and PD-1. This allows for prediction of tumor development and guidance of medication before treatment, evaluation of treatment efficacy after treatment, and guidance of subsequent treatment plans.

[0025] Immune cells are a common term for white blood cells, including lymphocytes and various phagocytes. They specifically refer to lymphocytes that can recognize antigens and produce specific immune responses; lymphocytes are the basic components of the immune system. Lymphocytes include T lymphocytes (CD3+), B lymphocytes, and NK cells, with T cells being the main component. CD3+ lymphocytes represent all T lymphocytes. CD4+ T lymphocytes refer to CD3+CD8+ cytotoxic T lymphocytes. CD8+ T cell infiltration is a tumor-related marker, and decreased CD8+ T cell infiltration is one of the predictors of poor prognosis.

[0026] In any of the above-mentioned preferred embodiments, step two involves dispersing the tumor tissue sample into single cells using eddy current centrifugal force. The eddy current centrifugal force described in this invention includes generating eddies in the solution containing the tumor tissue sample (or tumor cells) using an instrument. These eddies drive the solution to generate axial flow and shear rate. Under the action of the solution's liquid shear force, the tumor tissue sample (or tumor cells) overcomes the intercellular bonding forces and disperses into a single-cell suspension. The instrument for generating the eddies described in this invention includes, but is not limited to, a turbine oscillator and a magnetic stirrer, such as the Vortex-Genie2 vortex oscillator. The magnetic stirrer is preferably a micro-volume high-speed magnetic stirrer, such as the AMO series high-speed magnetic stirrer or the VELP micro-titering magnetic stirrer. The selected magnetic stirrer rotor can be a micro-volume PTFE magnetic stir bar (imported from the UK), with a length*diameter of 8*1.5 mm or 7*2 mm, or a micro-volume rotor adapted to the volume of the container used for cell processing. In existing technologies, single-cell suspensions used for flow cytometry are mostly obtained through enzymatic digestion with trypsin or other enzymes. This process damages the cell membrane and disrupts the distribution and quantity of proteins on the cell surface, leading to inaccurate test results. Tumor cells, compared to normal cells, have weaker intercellular connections. This invention utilizes the shear force (mechanical force) generated by eddy currents to disrupt these intercellular connections, thereby dispersing tumor tissue samples into individual tumor cells without damaging the cell membranes and surface proteins. The preferred eddy current centrifugal force is 2000-3000 rpm. This technical solution can be achieved using a vortex oscillator or a micro-magnetic stirring device capable of reaching speeds of 3000 rpm.

[0027] Preferably, the tumor tissue sample has a volume of 10-20 mm². 2 The tissue block. Further, in a preferred embodiment of the invention, the preferred tissue block volume is a tumor tissue block of 4mm x 4mm.

[0028] In any of the above-mentioned preferred embodiments, in step three, PD-L1 positive cells, PD-L1 negative cells, EGFR / ALK positive cells, and peripheral blood mononuclear cells (PBMCs) are mixed in a 1:1:1:1 ratio to prepare a control cell system. Flow cytometry detection of solid tumors differs from the detection of hematologic malignancies. In hematologic malignancies, some negative cells can be used as internal controls to help distinguish different cell populations. However, flow cytometry detection of solid tumors cannot use internal control cells and FSC / SSC methods for cell population differentiation, which is a challenge in flow cytometry detection of solid tumors. This invention adds external cells as controls, thus solving this challenge. Cells from the control system were added to single-tube control systems, named control, EGFR, ALK, CD45, PD-L1, and cell cycle, respectively. The control tube served as the negative control without antibody incubation. The EGFR, ALK, CD45, PD-L1, and cell cycle tubes were incubated with antibodies. A template was created for the flow chart, and compensation calculations were performed on the unincubated antibody control cells. The unincubated antibody control tubes were flow charted using FSC-A and SSC-A graphs, with voltage adjusted to center the cells in the FSC-A and SSC-A graphs, ensuring cells in the negative positions of each channel. A mixture of the control tubes and the single-tube control tubes was flow charted, cells were selected in the FSC-A and SSC-A graphs, and the voltage of each channel was adjusted to clearly separate the negative and positive peaks. The compensation value for each fluorescence channel was calculated. The calculated compensation values ​​were then used for flow chart analysis.

[0029] In this method, unincubated antibody control tubes were loaded onto the FSC-A and SSC-A graphs. The voltage was adjusted to position the cells in the center of the FSC-A and SSC-A graphs, with cells in each channel positioned in negative locations. The purpose of this voltage adjustment step is to clearly define the position of negative cell populations in each channel, making the separation of positive and negative cell populations distinct. Then, a compensation value was calculated to fix the position of positive cell populations, ensuring controllability of the entire experiment for positive cell analysis and guaranteeing the reliability and reproducibility of each analysis. Although voltage adjustment is a routine operation in flow cytometry, in current techniques, the purpose of voltage adjustment is to maintain cells in appropriate positions on the FSC-A and SSC-A graphs. This prevents cells from overflowing the signal-receiving boundary due to excessively high voltage or from being crowded together due to excessively low voltage, making them difficult to distinguish. This method of voltage adjustment mostly relies on experience, and the position of positive cell populations is not fixed. Determining whether a group of cells is positive requires judging the position of negative cell populations in the same graph. In the prior art, the compensation value is adjusted by using microspheres with different fluorescence to simulate cells for experimental setup or by relying on experience to obtain the compensation value for positive and negative cell groups in the experimental image, which is different from the method of this application.

[0030] Preferably, the cells used to prepare the control cell system include: PD-L1 positive cells H441, PD-L1 negative epithelial cells, EGFR / ALK positive cells H2228, and peripheral blood mononuclear cells (PBMCs).

[0031] Preferably, the above-mentioned method involves using the flow cytometry according to the voltage and compensation values ​​specified in the template, selecting live cells on the FSC-A / SSC-A plot, removing adhesions on the cell cycle plot, and applying the separated non-adhesions to the cell cycle plot to distinguish between diploid and aneuploid cells. Aneuploid cells are then applied to the CD45 / SSC plot, indicating that CD45-negative aneuploid cells are tumor cells. However, in existing tumor detection technologies, flow cytometry is only used to detect hematologic malignancies, which can be detected using only CD45 / SSC gating. Currently, PCR cannot distinguish between tumors and non-tumor cells, and immunohistochemistry determines whether tissue is tumor or normal by observing the appearance of cells after staining, but it cannot detect live cells. In the analytical method of this invention, instead of the traditional CD45 / SSC gating method, a new analytical method is designed. This invention utilizes CD45 and euploid / diploid gating and designs positive and negative control cells for tumor analysis. Traditional CD45 / SSC gating is often used to detect hematologic malignancies. Blood samples contain fragmented cells, leukocytes, and oligocytic erythrocytes. Hematologic malignancy analysis primarily targets different types of leukocytes. Since all leukocytes express CD45, CD45 / SSC gating can be used to select leukocytes. The sample used in this experiment differs from a blood sample; it is a tumor tissue sample containing both tissue cells and blood cells. Tissue cells cannot be distinguished using CD45.

[0032] Preferably, the tumor cell population is applied to each channel to obtain the proportion of EGFR+, ALK+, and PD-L1+ tumor cells. Through experiments, the flow cytometry results and IHC results of the same tissue samples are compared to obtain cutoff values. Samples with cutoff values ​​higher than the cutoff value indicate that one or more detected tumor markers are clinically significant and can be used for the evaluation of tumor treatment drugs or treatment plans.

[0033] This invention also provides a kit for evaluating targeted therapy for solid tumors, used in any of the above evaluation methods, comprising the following fluorescently labeled antibody combinations: a fluorescently labeled antibody combination consisting of CD45-PC7, PD-L1-APC, EGFR-FITC, ALK-PE, and Cell Cycle Dye-BV421; or a fluorescently labeled antibody combination consisting of EGFR-AF488, ALK-PE, CD45-PC7, AF647-PD-L1, and Cell Cycle Dye; and a fluorescently labeled antibody combination consisting of CD45-percp, CD3-APC, CD4-PC7, CD8-AC7, and PD-1-FITC. Different fluorescently labeled antibodies and antibodies with different clone numbers exhibit varying sensitivity and specificity in actual detection. Different combinations of biomarkers and fluorescent antibodies significantly impact the sensitivity of experimental detection; inappropriate combinations can even lead to cell overlap during detection, preventing effective detection and differentiation. This invention, through repeated experiments to obtain suitable fluorescent antibodies and optimal dosages, can achieve more distinct cell clustering and facilitate easier result analysis.

[0034] This invention has the advantages of requiring fewer samples and shorter detection time. The required tissue block can be as small as 4mm × 4mm, and the detection time does not exceed 5 hours. At the same time, it can provide information on a variety of factors that are helpful for prognosis and treatment, including the quantification of tumor-infiltrating lymphocytes, the proportion of tumor cells that are positive for PD-L1, EGFR, and ALK expression, and the proportion of tumor cells that are positive for co-expression.

[0035] Definitions of FSC and SSC: Forward scatter (FSC) refers to the detection of light scattering signals at a small forward angle (0.5°–2.0°), which essentially reflects the size of the cell. 90° scattered light, also known as side scatter (SSC), refers to scattered light perpendicular to the laser beam-fluid flow plane, and its signal intensity can reflect information about some cell structures. Attached Figure Description

[0036] Figure 1 The preferred embodiment of the present invention establishes the detection results of unmarked samples in the control tube of the template.

[0037] Figure 2 The preferred embodiment of the present invention establishes the labeling results of the Alexa Fluor 647 on the control tube sample of the on-machine template.

[0038] Figure 3 The marking results of the BV421 sample on the control tube of the preferred embodiment of the present invention are established.

[0039] Figure 4 The preferred embodiment of the present invention establishes the Alexa Fluor488 labeling results for the control tube sample of the on-machine template.

[0040] Figure 5 The PE marking results of the control tube sample on the machine template are established for the preferred embodiment of the present invention.

[0041] Figure 6 The PE-Cy7 marking results of the control tube sample on the machine template were established for the preferred embodiment of the present invention.

[0042] Figure 7 The results of testing fresh tumor tissue samples are from the preferred embodiment of the present invention, 2.

[0043] Figure 8 The results of the EGFR antibody titer experiment are from the preferred embodiment of the present invention.

[0044] Figure 9 The preferred embodiment of the present invention is the PD-L1 antibody titer experiment.

[0045] Figure 10 The antibody combination detection results are from the preferred embodiment 3 of the present invention.

[0046] Figure 11 The results of screening control cell lines are shown in the preferred embodiment 4 of this invention.

[0047] Figure 12 The results of live cell detection are from the preferred embodiment 5 of the present invention.

[0048] Figure 13 The results of cell cluster dispersion state detection are from the preferred embodiment 5 of the present invention.

[0049] Figure 14 The preferred embodiment of the present invention, 5, distinguishes between the detection results of tissue cells and non-tissue cells.

[0050] Figure 15 The preferred embodiment 5 of the present invention is the detection result of euploidy and aneuploidy.

[0051] Figure 16 The results of detection of each antibody combination in preferred embodiment 5 of the present invention.

[0052] Figure 17 The preferred embodiment of the present invention, 6, shows the detection results of immune cells surrounding solid tumors. Detailed Implementation

[0053] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can implement it based on the description. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the following embodiments are all conventional methods.

[0054] Example 1

[0055] Establishment of templates for flow cytometry and establishment of compensation values ​​for single-tube antibodies:

[0056] 1. A control cell system was prepared by mixing PD-L1 positive cells (H441), PD-L1 negative epithelial cells, EGFR / ALK positive cells (H2228), and peripheral blood mononuclear cells (PBMCs) at a ratio of 1:1:1:1, with a cell suspension concentration of 1.0 x 10⁻⁶. 6 per ml.

[0057] 2. Prepare single-tube controls. Take 6 1.5ml enzyme-free EP tubes and name each EP tube according to the name of the antibody added to each tube: control, EGFR, ALK, CD45, PD-L1, and cell cycle. Except for the control tube, add 200ul of control system cells to each tube and 500ul of control system cells to the control tube.

[0058] 3. Add 1 ml of 2% BSA to each tube and incubate at room temperature for 3-10 minutes to block the incubation.

[0059] 4. Centrifuge at 300g-800g for 5-10 minutes at room temperature, carefully observing the location of the precipitate;

[0060] 5. Discard the supernatant with a syringe, leaving 50-100µl of supernatant to prevent the accumulation of sediment;

[0061] 6. Remove the control tube, add 2-5 μL of the corresponding antibody to each tube, gently pipette to mix, and incubate at room temperature in the dark for 30 min.

[0062] 7. Add 1 ml of 2% BSA to each tube except the control tube, mix well by pipetting, and incubate at room temperature for 5 min.

[0063] 8. Centrifuge at 300-800g for 5 minutes at room temperature, discard the supernatant, and be careful not to aspirate the precipitate;

[0064] 9. Wash again.

[0065] 10. Before starting the test, mix the 100µl cells in the control tube with the 100µl cells in each of the other tubes and add them to the test tubes for testing.

[0066] 11. Set up the flow cytometer template, use unincubated antibody control cells as negative control, and perform compensation calculations using the software provided with the flow cytometer (Becton, Dickinson and Company, BD).

[0067] The results are as follows Figure 1 As shown.

[0068] Using the FSC-A and SSC-A graphs, load the unincubated antibody control tubes onto the machine. Adjust the voltage so that the cells are positioned in the middle of the FSC-A and SSC-A graphs, with the cells in each channel in the negative position.

[0069] Cells were selected from the control and mixed tubes of each single tube on the machine, and the voltage of each channel was adjusted to make the negative and positive peaks of each channel clearly distinct. Figure 2-6 As shown, the compensation values ​​for each fluorescence channel are calculated using the built-in flow cytometry software (BDFACSCanto II). The calculated compensation values ​​are then applied to the instrument. (Voltage values ​​can be used routinely on the same instrument for a short period; for longer intervals of testing, this procedure needs to be repeated.)

[0070] Example 2

[0071] Steps for testing fresh lung cancer tissue samples:

[0072] Postoperative lung cancer tissue samples were placed in RIPM cell preservation solution and stored at 2-8°C.

[0073] Preparation of fresh lung cancer tissue sample suspension on ice

[0074] Place RIPM and fresh lung cancer tissue together in a clean container, cut into approximately 4mm × 4mm pieces, and place them into clean 1.5ml EP tubes. Use D-PBS or RIPM to pipette and collect as many residual cells as possible from the container, transferring them into the 1.5ml EP tubes. Vortex or use a magnetic stirrer and micro-rotor for stirring. Turn on the power and set the vortex or stirrer speed to 3000rpm. The centrifugal force generated by the vortex in the culture medium will create shear force, causing cells to detach from the tumor tissue. Centrifuge the obtained cells, discard the supernatant, and wash once with D-PBS. Stain with 0.4% trypan blue; dead cells will stain blue. Perform viable cell counting under a microscope. At least 1 × 10⁴ cells can be prepared from a 4×4mm tissue block. 6 Single cell suspension per ml.

[0075] Sample preparation:

[0076] 1. Prepare a single-cell suspension (1.0 × 10⁶ cells) from the lung cancer tissue suspension to be tested. 6cells / mL;

[0077] 2. Take 250 μL of the above cell suspension and 1 mL of D-PBS solution into a 1.5 mL centrifuge tube, and centrifuge at 300 g-800 g for 5-10 min at room temperature;

[0078] 3. Discard the supernatant, leaving 50-100 μL of liquid, being careful not to aspirate any cell pellet;

[0079] 4. Add 100-350 μL of cell fixative (which can protect the integrity of antigens on the cell membrane), gently pipette to resuspend the cells, and incubate at room temperature for 1 hour. The fixatives described in this invention are all commercially available products in the prior art, preferably LiquiPrep cell preservation solution.

[0080] Preparation of control system:

[0081] A control cell system was prepared by mixing PD-L1 positive cells (H441), PD-L1 negative epithelial cells, EGFR / ALK positive cells (H2228), and peripheral blood mononuclear cells (PBMCs) at a ratio of 1:1.

[0082] Antibody markers:

[0083] 1. Take 200 μL of control cells and add them to a 1.5 mL centrifuge tube. Add 1 mL of 2%-5% BSA solution to each sample cell suspension and control cells, and incubate at room temperature for 5 min.

[0084] 2. Centrifuge at 300-800g for 5-10 minutes at room temperature;

[0085] 3. Discard the supernatant, leaving 50-100 μL of liquid, being careful not to aspirate any cell pellet;

[0086] 4. Mix the antibodies EGFR-AF488 ALK-PE CD45-PC7 AF647-PD-L1 with 2%-5% BSA at a ratio of 2-5 μL to prepare an antibody mixture;

[0087] 5. Add 50-200 μL of the prepared antibody to each sample tube, including the control sample system, and gently pipette to mix.

[0088] 6. Incubate at room temperature in the dark for 20-30 minutes.

[0089] Cell staining:

[0090] 1. Add 1 mL of 2%-5% BSA to each of the above-incubated sample and control tubes, mix gently, and incubate at room temperature for 5-10 min;

[0091] 2. Centrifuge at 300-800g for 5-10 minutes at room temperature;

[0092] 3. Discard the supernatant, leaving 50-100 μL of liquid, being careful not to aspirate any cell pellet;

[0093] 4. Repeat the above steps once;

[0094] 5. Add 100 μL of 1 μg / mL cell staining working solution to each sample and control tube, gently pipette to mix, and incubate at room temperature in the dark for 30 min.

[0095] The incubated samples were then fed into the flow cytometer:

[0096] Following the voltage and compensation values ​​in the template of Example 1, live cells are selected on the FSC-A / SSC-A diagram, and adhesions are removed on the cell cycle diagram. The separated non-adhesions are then applied to the cell cycle diagram to distinguish between diploid and aneuploid cells.

[0097] When applied to the CD45 / SSC plot, CD45-negative aneuploid cells are tumor cells (e.g., Figure 7 (The green group of cells in the middle left);

[0098] By applying tumor cell populations to various channels, the proportions of EGFR+, ALK+, and PD-L1+ tumor cells can be obtained.

[0099] As shown in Figure 7.

[0100] By comparing the proportion of EGFR+ALK+PD-L1+ tumor cells detected by flow cytometry in the same sample with that detected by conventional immunohistochemistry, a cutoff value can be obtained. Samples with cutoff values ​​higher than the cutoff value, combined with other clinical trials, can help doctors and patients choose appropriate drugs or treatment plans.

[0101] The cutoff value is determined as follows: The same sample is compared using flow cytometry and the gold standard method. This invention uses approximately 20 samples, testing them separately using both flow cytometry and the gold standard method. Statistical analysis reveals that samples with an ALK index above a certain value in the flow cytometry results are considered positive in the gold standard, while those below this value are considered negative. This value is defined as the cutoff value. Similarly, the cutoff values ​​for other indicators are determined in the same way.

[0102] Example 3

[0103] Example 3 uses the same experimental method as Example 1 or 2, except that it provides a selection of antibody combinations used in the detection of this invention. For example... Figure 8 The results shown are from the EGFR antibody titer experiment, indicating that EGFR antibody can be used in doses ranging from 0.5 μL to 2 μL to obtain good experimental results. Figure 9 The PD-L1 antibody titer experiment demonstrates that PD-L1 antibody can be used in doses ranging from 0.5 μL to 2 μL, all of which yield good experimental results. Figure 10 The antibody combination detection results show that the antibody combination provided by this invention has obvious positive and negative peaks, which can effectively separate cells.

[0104] Example 4

[0105] Example 4 uses the same experimental method as Examples 1-3, employing flow cytometry to screen control cell lines. The results are as follows: Figure 11 As shown, three types of lung cancer epithelial cells, A549, H1299, and H2228, were selected by reviewing the literature. Experiments revealed that H2228 cells can simultaneously express both EGFR and ALK proteins, and H2228 cells were finally selected as EGFR / ALK positive cells.

[0106] Example 5

[0107] Detection of solid tumor markers

[0108] Control cell composition: PD-L1 positive control cells H4441, PD-L1 negative control cells Beas-2B, EGFR, ALK positive control cells H2228, and PBMCs, each cell type was 1.0 × 10⁻⁶. 6 Prepared by mixing 1:1 per 1 mL.

[0109] 1. Fresh tumor tissue preserved at RIPM (cell culture medium) for no more than 48 hours was used to prepare a tissue suspension using a tissue suspension preparer, and the entire process was performed on ice. 20 μL of the tissue suspension was taken, stained with 0.4% trypan blue, and viable cells were counted.

[0110] 2. Prepare a single-cell suspension of at least 1.0 × 10⁻⁶ cells from the patient sample. 6 cells / mL;

[0111] 3. Take 250 μL of the above cell suspension and 1 mL of D-PBS solution into a 1.5 mL centrifuge tube, and centrifuge at 300 g for 5 min at room temperature;

[0112] 4. Discard the supernatant;

[0113] 5. Add 100-350 μL of cell fixative and cell permeabilizing agent, gently pipette to resuspend the cells, and incubate at room temperature for 1 hour.

[0114] Antibody markers:

[0115] 1. Take 200 μL of control cells and add them to a 1.5 mL centrifuge tube. Add 1 mL of 2% BSA solution to each patient sample cell suspension and control cells, and incubate at room temperature for 5 min.

[0116] 2. Centrifuge at 100-350g for 5 minutes at room temperature;

[0117] 3. Discard the supernatant, leaving 50-100 μL of liquid, being careful not to aspirate any cell pellet;

[0118] 4. Prepare antibodies according to the following system (prepare immediately before use).

[0119]

[0120] 5. Add 100 μL of the prepared antibody to each sample tube, including the control tube, and gently pipette to mix.

[0121] 6. Incubate at room temperature in the dark for 30 minutes.

[0122] Cell staining:

[0123] 1. Add 1 mL of 2-5% BSA to each of the above-incubated sample and control tubes, mix gently, and incubate at room temperature for 5-10 min;

[0124] 2. Centrifuge at 300-800g for 5-10 minutes at room temperature;

[0125] 3. Discard the supernatant, leaving 50-100 μL of liquid, being careful not to aspirate any cell pellet;

[0126] 4. Repeat the above steps once;

[0127] 5. Add 100 μL of 1 μg / mL Cell Cycle Dye staining working solution to tube 1 of each sample and the control tube, mix gently by pipetting, and incubate at room temperature in the dark for 20-30 min.

[0128] 6. The analysis was performed with a PD-L1 cutoff value of 5%, an EGFR cutoff value of 5%, and an ALK cutoff value of 6%.

[0129] like Figure 12 The image shows the results of a live cell detection test. The black dots outside the circled area represent dead cells and debris. Figure 13 As shown, the method provided by this invention allows tumor cell clusters to be dispersed into single cells. Figure 14 As shown, the green area represents CD45- tissue cells, and the blue area represents CD45+ non-tissue cells, thus distinguishing between tissue cells and non-tissue cells in the obtained cell suspension. Figure 15 The results are for detecting euploidy and aneuploidy. For example... Figure 16The results are for the detection of EGFR, ALK, and PD-L1. In this application, the cutoff value is defined as follows: when the proportion of tumor cells expressing a certain biomarker exceeds a certain percentage, the sample is considered positive for that biomarker. This certain percentage is the cutoff value, which is calculated. Specifically, in this embodiment, the PD-L1 cutoff value is calculated when the proportion of tumor cells expressing PD-L1 exceeds 5% of all tumor cells, indicating that the sample is PD-L1 positive. Similarly, the EGFR cutoff value is calculated when the proportion of tumor cells expressing EGFR exceeds 5% of all tumor cells, indicating that the sample is EGFR positive; the ALK cutoff value is calculated when the proportion of tumor cells expressing ALK exceeds 6% of all tumor cells, indicating that the sample is ALK positive. The calculation method for the cutoff value is as described in Example 2 and will not be detailed here.

[0130] Example 6

[0131] Tumor microenvironment detection

[0132] Fresh tumor tissues preserved at low temperature in RIPM (cell culture medium) for no more than 48 hours were prepared into tissue suspensions using a tissue suspension preparer, and the process was always carried out on ice.

[0133] 1. Centrifuge the tissue suspension, wash once with PBS, then stain with 0.4% trypan blue and perform viable cell counting.

[0134] 2. Prepare a single-cell suspension of patient samples with a density of at least 1.0 × 10⁶ cells / mL;

[0135] 3. Take 250 μL of the above cell suspension and 1 mL of D-PBS solution into a 1.5 mL centrifuge tube, and centrifuge at 300 g-600 g for 5-10 min at room temperature;

[0136] 4. Discard the supernatant;

[0137] 5. Add 250 μL of cell fixation solution, gently pipette to resuspend the cells, and incubate at room temperature for 1 hour.

[0138] 6. Add 1 mL of 2-5% BSA solution and incubate at room temperature for 5-10 min;

[0139] 7. Centrifuge at 300-600g for 5-10 minutes at room temperature;

[0140] 8. Discard the supernatant, leaving 50-100 μL of liquid. Be careful not to aspirate any cell pellet.

[0141] 9. Prepare antibodies according to the following dosage (prepare immediately before use).

[0142]

[0143] 10. Add 100 μL of the prepared antibody and gently pipette to mix.

[0144] 11. Incubate at room temperature in the dark for 20-30 minutes.

[0145] 12. Add 1ml of PBS, wash once, and then run on the instrument.

[0146] 13. Analyze immune cells in the microenvironment.

[0147] like Figure 17 This is the analysis result of immune cells surrounding solid tumors, where the immune cell analysis does not include cutoff values. Figure 17 The expression status of CD4+PD-1+ and CD8+PD1+ in immune cells surrounding solid tumors was shown.

[0148] Example 7

[0149] Example 7 uses the same experimental method as Examples 1-6. Example 7 provides specific cutoff data, as shown in Table 1 (in the table, % x 45neg refers to the proportion of 45-negative cells).

[0150] The cutoff value is obtained by comparing the flow cytometry results of the same tissue samples with the IHC results. Samples with a cutoff value higher than the cutoff value indicate that one or more tumor markers are clinically significant. A positive flow cytometry result for a marker with a cutoff value higher than the cutoff value is clinically significant and can be used to evaluate tumor treatment drugs or regimens.

[0151] Table 1

[0152]

[0153]

[0154] As shown in the table, compared with IHC detection results, the overall concordance rate for EGFR and ALK was 100%, and the overall concordance rate for PD-L1 was 89%. The flow cytometry method provided in this application, compared with immunohistochemistry, demonstrates significant consistency in the results of the same experiment. The high concordance rates of 100% and 89% indicate that the flow cytometry method provided by this invention can replace traditional immunohistochemistry and obtain accurate detection results.

Claims

1. A method for detecting targeted markers of solid tumors, comprising the detection of molecular markers for targeted therapy of solid tumors and the detection of the solid tumor microenvironment, the detection method comprising the following steps: Step 1: Collect fresh tumor tissue samples; Step 2: Prepare a single-cell suspension from the tumor tissue sample to be tested; Step 3: Establish a flow cytometry model; Step 4: Detect molecular markers for targeted therapy of solid tumors using flow cytometry; Step 5: Analyze the solid tumor microenvironment using flow cytometry; The order of steps four and five is not important; In step four, the molecular group of molecular markers for targeted therapy of solid tumors includes: CD45, PD-L1, EGFR, ALK, and CellCycle Dye; In step three, PD-L1 positive cells, PD-L1 negative cells, EGFR / ALK positive cells, and peripheral blood mononuclear cells (PBMCs) are mixed in a 1:1:1:1 ratio to prepare a control cell system. The control cells are then added to separate single-tube control systems, named control, EGFR, ALK, CD45, PD-L1, and cell cycle, respectively. The control tube serves as a negative control without added incubation antibody. Other control tubes include EGFR, ALK, CD45, PD-L1, and cell cycle tubes. The antibody was incubated in cycle 1; an instrument template was established, and compensation calculations were performed on the unincubated antibody control cells for negative control; the unincubated antibody control tubes were processed using FSC-A and SSC-A graphs, and the voltage was adjusted so that the cells were in the middle of the FSC-A and SSC-A graphs, and the cells in each channel were in the negative position; the control tubes were mixed with the individual tubes, and cells were selected in the FSC-A and SSC-A graphs, and the voltage of each channel was adjusted so that the negative and positive peaks of each channel were clearly separated, the compensation value of each fluorescence channel was calculated, and the calculated compensation value was processed.

2. The detection method as described in claim 1, characterized in that, In step five, immune cells surrounding solid tumors are detected to assess the solid tumor microenvironment. The molecular composition of immune cells surrounding solid tumors includes CD3, CD4, CD8, CD45, and PD-1.

3. The detection method as described in claim 1, characterized in that, Step two uses vortex centrifugal force to disperse the tumor tissue sample into single cells.

4. The detection method as described in claim 3, characterized in that, Tumor tissue samples with a volume of 10-20 mm 2 The organizational blocks.

5. The detection method as described in claim 1, characterized in that, The cells used to prepare the control cell system included: PD-L1 positive cells H441, PD-L1 negative epithelial cells, EGFR / ALK positive cells H2228, and peripheral blood mononuclear cells (PBMCs).

6. The detection method as described in claim 5, characterized in that, Follow the voltage and compensation values ​​in the template to run the computer. Select live cells on the FSC-A / SSC-A diagram, remove adhesions on the cell cycle diagram, and apply the separated non-adhesions to the cell cycle diagram to distinguish between diploid and aneuploid cells. Apply aneuploid cells to the CD45 / SSC diagram, and CD45-negative aneuploid cells are tumor cells.

7. The detection method according to any one of claims 1-6, characterized in that, The tumor cell population was applied to various channels to obtain the cutoff value, which was the proportion of EGFR+, ALK+, and PD-L1+ tumor cells. Samples with cutoff values ​​higher than the cutoff value were used to evaluate tumor treatment drugs or treatment plans.

8. A kit for detecting targeted markers in solid tumors, employing the detection method according to any one of claims 1-7, comprising the following fluorescently labeled antibody combinations: a fluorescently labeled antibody combination consisting of CD45-PC7, PD-L1-APC, EGFR-FITC, ALK-PE, and CellCycleDye-BV421, or a fluorescently labeled antibody combination consisting of EGFR-AF488, ALK-PE, CD45-PC7, AF647-PD-L1, and Cell CycleDye; and a fluorescently labeled antibody combination consisting of CD45-percp, CD3-APC, CD4-PC7, CD8-AC7, and PD-1-FITC.

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