CN114646584A

CN114646584A - Detection method of solid tumor targeting index and reagent combination thereof

Info

Publication number: CN114646584A
Application number: CN202011502574.0A
Authority: CN
Inventors: 赵子慧
Original assignee: Tianjin Kangshengda Medical Laboratory Co ltd
Current assignee: Tianjin Kangshengda Medical Laboratory Co ltd
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2022-06-21

Abstract

The invention provides a detection method of a solid tumor targeted index and a reagent combination thereof, which comprises the detection of a solid tumor targeted therapeutic molecular marker and the detection of a solid tumor microenvironment, wherein 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 cytometer on-machine model; detecting the solid tumor targeted therapeutic molecular marker by using flow cytometry; detecting a solid tumor microenvironment using flow cytometry. The invention needs less samples for detection, has short detection time, and can provide information about a plurality of factors which are helpful for prognosis and treatment, including the quantification of tumor infiltrating lymphocytes, the proportion of tumor cells positive for expression of PD-L1, EGFR and ALK and the proportion of tumor cells positive for co-expression.

Description

Detection method of solid tumor targeting index and reagent combination thereof

Technical Field

The invention relates to detection of solid lung cancer tumor cell markers and driving genes, and particularly provides a detection method of lung cancer tumor targeting indexes and a reagent combination for detecting tumors and microenvironment markers thereof in the detection method.

Background

Lung cancer is the most common malignant tumor with the highest morbidity and mortality worldwide, and the majority of lung cancer is non-small cell lung cancer, accounting for about 80% -85%. Although the proportion of middle and late stage tumors is reduced in the first diagnosis result of newly-increased lung cancer patients with the enhancement of health consciousness of residents and the application of early screening technologies such as spiral CT and the like, the late stage treatment effect is poor and the prognosis is poor due to tumor heterogeneity, and the relative survival rate in 5 years is only 16.1%. How to more effectively prolong the survival rate of lung cancer patients and reduce the death rate of the lung cancer patients is always a focus of attention in lung cancer treatment. Non-small cell lung cancer (NSCLC) patients of the same stage, with different tissue subtypes, different driver genes, will produce different therapeutic effects and prognosis for the same treatment modality. For most patients with non-small cell lung cancer, surgical removal of tumors remains the treatment of choice. In patients with advanced non-small cell lung cancer, the focus usually has metastasized, and the surgery cannot achieve the expected therapeutic effect. By accurately staging the advanced non-small cell lung cancer, determining the subtype, analyzing the molecular lung cancer marker and detecting the driving gene and selecting a proper treatment means and a targeted drug, the prognosis of the non-small cell lung cancer patient can be effectively improved, and the survival rate is improved.

As the development of the platinum-containing chemotherapeutic drug for treating NSCLC enters a bottleneck period, the macromolecule targeting treatment mode represented by an epidermal growth factor tyrosine kinase inhibitor remarkably prolongs the progression-free survival period of the late NSCLC positive for the driving gene, but the instability of the driving gene related to the development of the NSCLC determines that the targeted drug is difficult to cure the tumor. Immunotherapy, a promising treatment for tumors, has multiplied the 5-year survival rate of advanced NSCLC, despite the poor prognosis in some patients.

Activation of T cells of the immune system requires at least two signals: the first signal is that a particular peptide presented by the antigen presenting cell APC bound to a Major Histocompatibility Complex (MHC) molecule is recognized by the T cell antigen receptor TCR; the second signal is the linkage of a costimulatory molecule (e.g., CD28, etc.) to its selective ligand B7 on the APC. Two signals are lack of one signal and can cooperate with each other to promote T cell activation, secrete cell factors and play immune function. T cell activation is not without limitation, and when T cells are activated for a certain period of time, the inhibitory costimulatory molecule PD-1 expressed on the activated T cells inhibits T cell activation, mediates T cell apoptosis. The positive and negative co-stimulatory molecule signals determine the strength and freedom of immune response, and the tumor immunotherapy refers to the control and killing of tumor cells by stimulating or mobilizing the immune system of the body and enhancing the anti-tumor immunity of the tumor microenvironment.

Epidermal Growth Factor Receptor (EGFR), a receptor type tyrosine kinase, is expressed on the cell membrane. Under normal conditions, the extracellular end of EGFR is combined with ligand to form a dimer, the intracellular end is combined with ATP to generate phosphorylation, and then a plurality of downstream signal channels, such as signal channels of PKC, JAK, ERK and the like, are activated, and a plurality of physiological processes of cell growth, differentiation, development and the like are regulated. EGFR overexpression and mutation can cause activation of EGFR structural domain, abnormally activate downstream signal channels and promote malignant proliferation of tumor cells. EGFR is considered to be one of the effective targets for treating cancer, and many anti-EGFR inhibitor drugs including EGFR monoclonal antibodies cetuximab, nituzumab, panitumumab, and small molecule Tyrosine Kinase Inhibitors (TKI) erlotinib, gefitinib, erlotinib, etc. have been developed and studied so far.

The Anaplastic Lymphoma Kinase (ALK) gene is considered a strong cancer driver gene because rearrangement of the ALK gene is detected in both hematological and solid malignancies, including anaplastic large cell lymphoma, myofibrobronchial tumors, and NSCLC, and is involved in the development and progression of tumors. The ALK gene is mainly expressed in the embryonic stage, promotes the proliferation of nerve cells and plays a role in the development process of the brain and the peripheral nervous system. When the nervous system has developed completely, it usually enters a dormant state, and other cells in the human body do not substantially express ALK, which is usually activated by chromosomal rearrangement.

Tumors are located in tumor microenvironments, including tumors, immune cells and stroma, which interact together to determine the fate of a particular cancer. Treatment of tumors therefore requires sophisticated medicine to collect and evaluate all information related to the tumor. At present, detection methods of important molecular targets PD-L1, EGFR, ALK and the like for treating lung cancer comprise immunohistochemistry, sequencing, PCR, FISH and the like. The detection techniques have the disadvantages of multiple steps, large amount of required samples, high detection cost and the like. The traditional methods are all single detection, and a single detection can only provide relatively single detection information, so that the detection of multiple tumor biomarkers is difficult to complete.

The flow cytometry is to prepare single cell suspension of the sample to be measured, stain the single cell suspension with specific fluorescent dye, set the single cell suspension in a sample tube, and discharge the single cell suspension. The stained single cells are excited by the incident laser light when passing through the laser focusing area, and fluorescence with a specific wavelength is generated. The state, type, DNA content and the like of the cells can be distinguished through analyzing signals such as fluorescence, light scattering, light absorption or cell resistance and the like collected by the instrument; flow cytometry combined with monoclonal antibodies allows for the quantitative detection of cell surface and intracellular antigens, oncoproteins, and membrane receptors. Clinically, the flow detection is used for detecting the immune state and the micro-focus residue of an organism, evaluating the treatment effect and the prognosis of a medicament, and the like. The tumor is divided into solid tumor and non-solid tumor clinically, solid tumor and tangible tumor can be called solid tumor by clinical examination such as X-ray radiography, CT scanning and B-ultrasonic or palpation of the tangible tumor, and the non-solid tumor, X-ray, CT scanning, B-ultrasonic and palpation of the tangible tumor such as leukemia in blood disease belongs to the non-solid tumor. In the prior art, most samples detected by flow cytometry are blood samples, cell samples cultured in vitro and the like, and are less used for detecting tissue type samples. Solid tumor samples are subjected to flow detection, and single cell suspensions are generally obtained by physical grinding or enzyme digestion, but the methods can damage the cell surface structure, so that the composition and the quantity of surface proteins are damaged, the single cell suspension yield is low, and the detection accuracy and sensitivity are influenced, so that the flow cytometry is difficult to be used for detecting clinical solid tumors.

Disclosure of Invention

In order to overcome the defects, the invention provides an evaluation method of solid tumor targeted therapy, which comprises the detection of a molecular marker of the solid tumor targeted therapy and the detection of immune cells around a solid tumor, and the evaluation method comprises the following steps:

the method comprises the following steps: collecting a fresh tumor tissue sample;

step two: preparing a tumor tissue sample to be detected into a single cell suspension;

step three: establishing a flow cytometer on-machine model;

step four: detecting the solid tumor targeted therapeutic molecular marker by using flow cytometry;

step five: detecting immune cells around the solid tumor by using flow cytometry;

wherein, the sequence of the step four and the step five is not divided into sequence.

In the prior art, the treatment evaluation of the tumor is only limited to the detection of the tumor cell, and the evaluation method provided by the application not only detects the marker of the tumor cell, but also detects the immune marker in the microenvironment where the tumor cell is located. Thereby allowing a systematic and comprehensive evaluation of the tumor drug and/or treatment regimen. The solid tumor microenvironment refers to an immune microenvironment in which solid tumor cells are located, and the solid tumor cells comprise tumor cells and non-tumor cells, and the non-tumor cells form the microenvironment of the tumor cells. Furthermore, the solid tumor microenvironment of the invention is used for detecting immune cells around tumor tissues obtained in operation, and aims to judge the microenvironment of the tumor around a human body. The detection of the microenvironment of the solid tumor according to the present invention preferably means detection of immune cells surrounding the solid tumor, and the detection of immune cells surrounding the tumor according to the present invention preferably further detects percentage change of CD4+, CD8+ cells, and/or PD-1 change on related immune cells. The microenvironment is different from the traditional microenvironment, and the solid tumor microenvironment does not detect the change of immune cells in the whole peripheral blood, but reflects the microenvironment of the solid tumor more finely according to the expression condition of the immune cells at the position of the tumor. The evaluation provided by the application can detect the change of the tumor cells before and/or after treatment and the change of the microenvironment in which the tumor cells are positioned, including but not limited to the types of immune cells in the microenvironment, the types and the expression content of cytokines expressed by each immune cell. The invention discloses a targeted therapy molecular biomarker for detecting solid tumors by a flow method.

Preferably, in step four, the molecular group of the molecular markers for the solid tumor targeted therapy comprises: CD45, PD-L1, EGFR, ALK and Cell Cycle Dye, wherein the EGFR and the ALK are the most common driving genes for lung cancer, and account for the vast majority of Asian non-small Cell lung cancer patients. PD-L1 is an immunological screening point for determining whether a patient is eligible for immunotherapy and prognosis.

cell cycle dye can detect whether a cell is aneuploid or diploid, and together with CD45, distinguish solid tumor cells from non-tumor cells. The invention firstly applies the combination of euploid detection and CD molecule mode to the flow type solid tumor detection, namely the combination application of CD45 and Cell Cycle Dye distinguishes tumor cells from non-tumor cells. The detection mode is different from that of the flowing blood tumor, and is mainly distinguished by forward scattering FSC and side scattering SSC combined with CD molecules.

Through the detection of the expression levels of the tumor markers PD-L1 and the driving genes EGFR and ALK, the method helps clinicians to judge the disease progress, select drugs and judge the prognosis effect of tumor patients, and achieves the purpose of auxiliary diagnosis. In the prior art, whether two genes EGFR and ALK in a tumor tissue sample have pathogenic mutation can be clinically detected by a PCR (polymerase chain reaction) or first-generation sequencing and fluorescence in situ hybridization method, the expression levels of three index proteins in the tumor tissue can be measured by an immunohistochemical method, and then the disease progress condition of a patient can be judged. The method can only obtain the detection result of a single index in a single sample experiment, namely, only one index can be detected in each experiment of one tissue sample, and the detection results of the three indexes can be simultaneously obtained in a single experiment of one tissue sample. Compared with an immunohistochemical detection method, the flow detection method is more accurate in detection method, can avoid the interpretation of subjective factors on the positivity and negativity of an experiment, and can give the proportion of tumor cells expressing PD-L1, ALK and EGFR in an experimental sample. By the method, three markers of PD-L1, EGFR and ALK, CD45 and Cell Cycle Dye are detected simultaneously by flow cytometry, and the expression levels of PD-L1, EGFR and ALK proteins in a tumor sample are reflected according to the detection result. The invention adopts flow cytometry for detection, only uses one tissue sample for detecting the molecular biomarker of the tumor at one time, and has the advantages of high accuracy, short detection time and less adopted tissue samples.

The CD45 molecule is expressed on all leukocytes and is called Leukocyte Common Antigen (LCA). CD45 is composed of a kind of transmembrane protein with similar structure and larger molecular weight, is widely present on the surface of leucocytes, and the cytoplasmic segment of the protein has the function of protein tyrosine phosphatase, can lead tyrosine on substrates P56lck and P59fyn to be dephosphorylated and activated, and plays an important role in the information transmission of cells. CD45 is a key molecule for signal transduction on cell membranes, has important significance in the developmental maturation, function regulation and signal transmission of lymphocytes, and the distribution of CD45 can be used as a classification marker of certain T cell subsets. In the present invention, CD45 is used to distinguish tumor cells in solid tumors, i.e., CD 45-negative group of cells is a tumor cell group.

The tumor marker PD-L1 is a ligand co-inhibiting Programmed death receptor PD-1, also known as Programmed cell death 1ligand, and is expressed on many tumor cells including non-small cell lung cancer. And the PD-L1 which is greatly expressed on the surface of the tumor cell is combined with PD1 on the T cell, so that the T cell cannot recognize the tumor cell, and the T cell is prevented from being subjected to 'killing' by the T cell, so that the strong immune response is avoided, and the growth and the proliferation are maintained. Based on the method, the immunotherapy of the tumor is to prevent the combination of PD-L1 on the tumor cell and PD1 on the T cell by competitive binding of the targeted drug to PD-L1 on the tumor cell or PD-1 on the T cell, so that the normal activation of the immune response of the tumor patient is ensured. NCCN guidelines have listed PD-L1 as a biomarker for non-small cell lung cancer as an immune screening point for necessary assessment in patients with advanced stage nsclc, and IHC should detect expression levels of PD-L1 in tumor tissues that are not less than 50%. The low expression of PD-L1 in tumor tissues is one of the prediction markers of poor curative effect of immunotherapy.

EGFR is a driver of non-small cell lung cancer, and overexpression of EGFR has been shown to be associated with prognosis and metastatic spread of various cancers, with most EGFR overexpression being significantly associated with lymph node metastasis and more advanced stages of pathology. In lung cancer, overexpression of EGFR protein and EGFR gene amplification are more common in adenocarcinoma, EGFR gene is overexpressed in 70% of adenocarcinoma patients, and EGFR gene expression levels are significantly different in non-small cell lung cancer patients at different stages. Non-small cell lung cancer studies have found that EGFR expression is associated with decreased survival, and EGFR overexpression in advanced NSCLC patients is associated with cancer cell survival, metastasis, invasion, and chemotherapy resistance, often implying poor survival and poor prognosis.

ALK is also a driver gene of non-small cell lung cancer, and ALK rearrangements occur in about 1% -7% of non-small cell lung cancer patients, and a variety of ALK rearrangements have been clinically found, with ALK-EML4 being the most common among non-small cell lung cancers. Methods for detecting ALK rearrangement mainly include Fluorescence In Situ Hybridization (FISH), Immunohistochemistry (IHC), Polymerase Chain Reaction (PCR), and the like. The FISH can specifically and sensitively detect ALK rearrangement, is the most common method for clinically detecting ALK fusion genes at present, PCR can sensitively detect known ALK rearrangement, and the accuracy and sensitivity of IHC for detecting the expression of the ALK rearrangement depend on antibodies to a great extent, so the FISH is usually used for primary screening of ALK gene rearrangement. The 18-year Chinese non-small cell lung cancer treatment guideline uses VENTANA ALK (D5F3) IHC detection kit as the clinical preferred conventional diagnosis method of ALK positive NSCLC, so as to be different from the initially screened conventional IHC method, but the diagnosis needs to be confirmed by either FISH or VENTNANA ALK, PCR after the initial screening of the conventional IHC.

Preferably, in any of the above steps, the combination of molecules for detecting solid tumor microenvironment in step five comprises CD3, CD4, CD8, CD45 and PD-1. The present invention has the advantages of detecting the immune environment around solid tumor and the expression of immune cell, especially the detection of markers CD3, CD4, CD8, CD45 and PD-1, predicting tumor development and guiding medicine before treatment, evaluating the treatment effect after treatment and guiding the subsequent treatment scheme by using flow cytometry.

Immune cells are commonly known as leucocytes, and comprise lymphocytes, various phagocytes and the like, and particularly refer to lymphocytes which can recognize antigens and generate specific immune response, and the like, and the lymphocytes are basic components of the immune system. Lymphocytes include T lymphocytes (CD3+), B lymphocytes, NK cells, among which T cells are the major component of lymphocytes. Lymphocytes of CD3+ represent whole T lymphocytes. CD4+ T lymphocytes refer to the cytotoxic T lymphocytes of CD3+ CD8 +. Wherein CD8+ T cell infiltration is a related marker of tumor, and the reduction of CD8+ T cell infiltration is one of poor prognosis prediction markers.

Preferably, in any of the above steps, the tumor tissue sample is dispersed into single cells by vortex centrifugal force. The eddy centrifugal force comprises that an instrument generates eddy in a solution in which a tumor tissue sample (or tumor cells) is positioned, the eddy drives the solution to generate axial flow and shearing rate, and the tumor tissue sample (or tumor cells) overcomes intercellular connection force under the action of the shearing force of the solution liquid to be dispersed into single cell suspension. The Vortex generating apparatus of the present invention includes, but is not limited to, a turbine oscillator and a magnetic stirrer, such as Vortex oscillator of Vortex-Genie2, preferably a micro high speed magnetic stirrer, such as AMO series high speed magnetic stirrer, VELP microtitre magnetic stirrer, the magnetic stirrer rotor selected may be a micro tetrafluoro magnetic stirrer (uk import), length x diameter: 8 x 1.5MM or 7 x 2MM or a micro rotor adapted to the volume of the container used for cell processing. In the prior art, a single cell suspension for flow cytometry is mostly obtained by means of digestion with enzymes such as trypsin, and in the process, cell membranes are damaged, and the distribution and the quantity of proteins on the cell surfaces are damaged, so that the detection result is inaccurate. Compared with normal cells, the tumor cells have weaker intercellular connection force, and the shearing force (mechanical force) generated by vortex flow can break intercellular connection of the tumor cells, so that the tumor tissue sample is dispersed into single tumor cells without breaking cell membranes and surface proteins of the tumor cells. The preferred vortex centrifugal force is 2000-3000 rpm. The technical scheme can be realized by a vortex oscillator or a micro magnetic stirring device with the speed of 3000 rpm.

Preferably, in any of the above cases, the tumor tissue sample has a volume of 10-20mm²The tissue mass of (1). Further in preferred embodiments of the present invention, a tumor tissue mass having a tissue mass volume of 4mmx4mm is preferred.

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 PBMC are mixed at a ratio of 1:1:1:1 to prepare control system cells. The solid tumor flow detection is different from the detection of the blood tumor, partial negative cells can be used as an internal control in the blood tumor detection to help distinguish each group of cells, and the flow detection of the solid tumor cannot be grouped by the internal control cells and the FSC SSC, which is also the difficulty of the flow detection of the solid tumor. The invention adds external cells as a control, thus solving the difficulty. Respectively adding cells of a control system into single-tube control tubes, wherein the single-tube control tubes are respectively named as control, EGFR, ALK, CD45, PD-L1 and cell cycle; wherein, the control tube is a negative control and does not add an incubation antibody, and the EGFR tube, the ALK tube, the CD45 tube, the PD-L1 tube and the cell cycle are used for antibody incubation; establishing an upper computer template, and performing compensation calculation on negative control non-incubated antibody io control cells; using an FSC-A graph and an SSC-A graph to machine an unincubated antibody Control tube, and adjusting the voltage to ensure that the cells are in the middle of the FSC-A graph and the SSC-A graph and the cells of each channel are in a negative position; and (3) operating the control tube and the mixing tube of each single tube, selecting cells on FSC-A and SSC-A graphs, adjusting the voltage of each channel to ensure that the negative peak and the positive peak of each channel are obviously grouped, and calculating the compensation value of each fluorescence channel. And (4) operating the machine according to the calculated compensation value.

Where the antibody Control tubes were not incubated on the FSC-A and SSC-A plots, the voltage was adjusted so that the cells were in the middle of the FSC-A and SSC-A plots, and the cells of each channel were in the negative position. The function and purpose of adjusting the voltage in this step are to make sure the position of the negative cell group in each channel map by adjusting the voltage, so that the grouping of the negative and positive cell groups in each channel is obvious, then calculate the compensation value, fix the position of the positive cell group, so that the whole experiment can control the analysis of the positive cell, and ensure the reliability and repeatability of each experimental analysis. Although voltage regulation is a routine operation of flow experiments, in the prior art, the voltage regulation is used for keeping cells at proper positions on FSC-A and SSC-A graphs and avoiding that the cells are extruded out of boundaries where an aggregate signal can be received due to too high voltage or the voltage is too low, so that different groups of cells are pressed together and are difficult to distinguish. In the prior art, the adjustment compensation value is adjusted through an on-machine experiment by using microsphere simulation cells with different fluorescence or by experience, so as to obtain a fixed compensation value of a negative and positive cell group of a group in an on-machine diagram, which is different from the method of the application.

Preferably, in any of the above cases, the cells from which the control cell system is prepared comprise: PD-L1 positive cells H441, PD-L1 negative epithelial cells, EGFR/ALK positive cells H2228 and peripheral blood mononuclear cells PBMC.

Any one of the above is preferably operated according to the voltage and compensation values in the operating template, selecting live cells on the FSC-A/SSC-A map, removing the cohesiveness in the cell cycle map, applying the separated non-cohesiveness to the cell cycle map, and distinguishing diploid cells from aneuploid cells; when the aneuploid cells were applied in a CD45/SSC map, the CD45 negative aneuploid cells were tumor cells. In the existing tumor detection technology, the flow cytometry is only used for detecting the blood tumor, and the detection of the blood tumor only needs to set a door of CD 45/SSC. At present, PCR cannot distinguish tumor from non-tumor, immunohistochemistry is to judge whether tumor tissue or normal tissue by looking at the tissue cell sample after staining, and living cells cannot be detected. In the analysis method of the invention, a traditional CD45/SSC gating method is not adopted, but a new analysis method is designed, the invention utilizes a CD45 and euploid diploid method to gate, and negative and positive control cells of the tumor are designed for analyzing and gating. The conventional CD45/SSC gate is mostly used for detecting hematological tumors, and the blood sample includes debris cells, white blood cells and red blood cells with few cells. Analysis of hematological tumors is mainly directed to different types of leukocytes, all of which express CD45, so that leukocytes can be selected by gating on CD 45/SSC. The sample used in the experiment is different from a blood sample, is a tumor tissue sample, comprises tissue cells and blood cells, and the tissue cells cannot be distinguished by CD 45.

In any of the above cases, it is preferable that the tumor cell population is applied to each channel to obtain the ratios of EGFR +, ALK +, and PD-L1 +. Through experiments, the cutoff value is obtained by comparing the flow type experiment result of the same tissue sample with the IHC experiment result, and the sample higher than the cutoff value means that one or more samples have clinical significance in detecting the positive tumor markers, so that the method can be used for evaluating tumor treatment medicines or treatment schemes.

The invention also provides a kit for the targeted therapy evaluation of solid tumors, which is used for the evaluation method of any one of the above methods, and comprises the following fluorescence labeled antibody combinations: a combination of fluorescence labeled antibodies consisting of CD45-PC7, PD-L1-APC, EGFR-FITC, ALK-PE and Cell Cycle Dye-BV421 or a combination of fluorescence labeled antibodies consisting of EGFR-AF488, ALK-PE, CD45-PC7, AF647-PD-L1 and Cell Cycle Dye; and a combination of fluorescently labeled antibodies consisting of CD45-percp, CD3-APC, CD4-PC7, CD8-AC7, and PD-1-FITC. The sensitivity and specificity of different fluorescence labeled antibodies and antibodies with different clone numbers in actual detection are different. The combination of different markers and different fluorescent antibodies has great influence on the sensitivity of experimental detection, even improper combination causes cells to overlap during detection and cannot be effectively detected and distinguished, the invention can obtain proper fluorescent antibodies and optimal using amount through repeated experiments, so that the grouping of cells is more obvious, and the result analysis is easier.

The invention has the advantages of less samples required for detection and short detection time, the required tissue block can be only 4mm multiplied by 4mm, the detection time does not exceed 5h, and simultaneously, the invention can provide information about various factors which are helpful for prognosis and treatment, including the quantification of tumor infiltration lymphocytes, the proportion of tumor cells positive for expression of PD-L1, EGFR and ALK and the proportion of tumor cells positive for co-expression.

The terms for FSC and SSC are defined as: the light scattering signal is detected at small forward angles (0.5-2.0), called Forward Scatter (FSC), which substantially reflects the size of the cell volume; the 90 ° scattered light is also called Side Scatter (SSC), and refers to scattered light perpendicular to the laser beam-liquid flow plane, and its signal intensity can reflect the information of the partial structure of the cell.

Drawings

FIG. 1 is a diagram illustrating the establishment of an unlabeled detection result for a sample in a template control tube according to a preferred embodiment 1 of the present invention.

FIG. 2 shows Alexa Fluor 647 labeling results of the control tube sample established on the computer template in the preferred embodiment 1 of the present invention.

FIG. 3 shows the result of establishing BV421 labeling on the upper template control tube sample in the preferred embodiment 1 of the present invention.

FIG. 4 shows the result of Alexa Fluor488 labeling of the sample from the control tube set up as the upper computer template in the preferred embodiment 1 of the present invention.

FIG. 5 shows the result of PE labeling on the control tube sample of the upper computer template in the preferred embodiment 1 of the present invention.

FIG. 6 shows the result of PE-Cy7 labeling on the control tube sample of the upper computer template set up in the preferred embodiment 1 of the present invention.

FIG. 7 shows the result of detecting fresh tumor tissue samples according to the preferred embodiment 2 of the present invention.

FIG. 8 shows the results of EGFR antibody titer assay in accordance with a preferred embodiment of the present invention.

FIG. 9 is a preferred embodiment of the invention 3PD-L1 antibody titer assay.

FIG. 10 shows the results of the antibody combination assay of preferred embodiment 3 of the present invention.

FIG. 11 shows the results of screening control cell lines in accordance with the preferred embodiment 4 of the present invention.

FIG. 12 shows the results of the detection of living cells in the preferred embodiment 5 of the present invention.

FIG. 13 shows the result of examining the state of dispersion of cell masses in accordance with preferred embodiment 5 of the present invention.

FIG. 14 shows the result of the test for differentiating tissue cells from non-tissue cells according to the preferred embodiment 5 of the present invention.

Fig. 15 shows the results of the detection of euploid and aneuploid in the preferred embodiment 5 of the present invention.

FIG. 16 shows the results of the detection of each antibody combination in the preferred embodiment 5 of the present invention.

FIG. 17 shows the detection results of the immune cells around the solid tumor in the preferred embodiment 6 of the present invention

Detailed Description

The present invention is further described with reference to specific examples, which enable one skilled in the art to practice the invention with reference to the description. However, the present invention is not limited to the following examples. In the following examples, unless otherwise specified, all the methods are conventional.

Example 1

Establishing a machine template by flow cytometry, and establishing a compensation value of a single-tube antibody:

1. PD-L1 positive cells H441, PD-L1 negative epithelial cells, EGFR/ALK positive cells H2228, and peripheral blood mononuclear cells PBMC were mixed at a ratio of 1:1:1 mixing to prepare cells of a control system, wherein the concentration of a cell suspension is 1.0x10⁶One per ml.

2. Preparing a single tube control, taking 6 1.5ml enzyme-free EP tubes, respectively adding the antibody name into each tube, and respectively naming each EP tube as control, EGFR, ALK, CD45, PD-L1 and cell cycle, adding 200ul of control system cells into each tube except the control tubes, and adding 500ul of control system cells into the control tubes;

3. adding 1ml of 2% BSA into each tube, incubating at room temperature for 3-10min, and sealing;

4. centrifuging at room temperature of 300-800g for 5-10min, and observing the position of the precipitate;

5. discarding the supernatant with a gun, and reserving 50-100ul of supernatant to prevent the precipitate from being absorbed;

6. removing the control tube, adding 2-5ul of corresponding antibody into each tube, gently blowing, beating and mixing, and incubating at room temperature in dark for 30 min;

7. removing control tube, adding 1ml 2% BSA per tube, blowing, mixing, and incubating at room temperature for 5min

8. Centrifuging at room temperature of 300-;

9. repeatedly washing once

10. Respectively mixing 100ul cells in a control tube before being loaded on a machine with 100ul cells in each tube, and adding the mixture into the upper machine tube;

11. loading the machine, establishing an upper machine template, carrying out negative control on an antibody control cell which is not incubated, and carrying out compensation calculation by using self-contained software of a flow cytometer (Becton, Dickinson and Company, BD for short);

the results are shown in FIG. 1.

The unincubated antibody Control tubes were machined on FSC-A and SSC-A plots, and the voltage was adjusted so that the cells were in the middle of the FSC-A and SSC-A plots, with the cells of each channel in the negative position.

The machine is respectively operated to control the mixing tube with each single tube, cells are selected on FSC-A and SSC-A graphs, the voltage of each channel is adjusted, so that negative peaks and positive peaks of each channel are obviously grouped, and as shown in figures 2 to 6, compensation values of each fluorescence channel are calculated by using self-contained flow analysis software (BD FACSCAnto II software). And (4) operating the machine according to the calculated compensation value. (the same instrument with the voltage value can be used conventionally in a short time, and the detection needs to be repeated again for a longer time)

Example 2

Detecting a fresh tissue lung cancer sample:

the lung cancer tissue sample after the operation is placed in RIPM cell preservation solution and preserved at the temperature of 2-8 ℃.

Fresh lung cancer tissue sample suspension preparation on ice

Putting RIPM and fresh lung cancer tissues into a clean container, cutting into small blocks of about 4mm multiplied by 4mm, putting into a clean 1.5ml EP tube, blowing and beating with D-PBS or RIPM, collecting the residual cells in the container as completely as possible, putting into a 1.5ml EP tube, shaking by a vortex oscillator or stirring by a magnetic stirrer and a miniature rotor, starting a power supply, wherein the rotation speed of the oscillator or the stirrer is 3000rpm, and generating shearing force by using centrifugal force generated by vortex of culture solution to enable the cells on the tumor tissues to fall off. The resulting cells were centrifuged, and the supernatant was discarded, followed by washing with D-PBS. Dead cells were stained blue with 0.4% trypan blue, microscopicCounting viable cells under the mirror, 4X 4mm tissue blocks, at least 1X 10⁶Single cell suspension per ml.

Preparing a sample to be detected:

1. preparing the lung cancer tissue suspension to be detected into single cell suspension of 1.0x10⁶Per mL;

2. taking 250 mu L of the cell suspension, 1mL of D-PBS solution and 1.5mL of a centrifuge tube, and centrifuging for 5-10min at room temperature at 300g-800 g;

3. the supernatant was aspirated away, leaving 50-100. mu.L of liquid, taking care not to aspirate the cell pellet;

4. add 100-. The fixing solution is a commercial product in the prior art, and is preferably LiquiPrep Lipu cell preservation solution.

Preparation of a control system:

PD-L1 positive cells H441 PD-L1 negative epithelial cells, EGFR/ALK positive cells H2228 and peripheral blood mononuclear cells PBMC were mixed at a ratio of 1:1 to prepare a control cell system.

Antibody labeling:

1. adding 200 mu L of control cells into a 1.5mL centrifuge tube, respectively adding 1mL of 2% -5% BSA solution into each cell suspension of the sample to be detected and the control cells, and incubating for 5min at room temperature;

2. centrifuging at room temperature for 5-10min at 800 g;

4. uniformly mixing the antibody EGFR-AF488 ALK-PE CD45-PC7 AF647-PD-L1 with 2% -5% BSA according to the proportion of 2-5ul to prepare an antibody mixture;

5. each sample comprises a control sample system, wherein 50-200 mu L of prepared antibody is added into a tube, and the mixture is lightly blown and uniformly mixed;

6. incubating at room temperature in dark for 20-30 min.

Cell staining:

1. adding 1mL of 2% -5% BSA into each incubated sample and the control tube, gently mixing, and incubating at room temperature for 5-10 min;

2. centrifuging at room temperature for 5-10min and at the temperature of 300-;

4. repeating the steps for 1 time;

5. adding 100 mu L of 1 mu g/mL cell staining working solution into each sample and each control tube, gently blowing, uniformly mixing, and incubating for 30min at room temperature in a dark place;

the incubated samples were run on a flow machine:

the cells were selected for viability on the FSC-A/SSC-A plot, the cohesiveness was removed in the cell cycle plot, and the isolated non-cohesiveness was applied to the cell cycle plot to distinguish diploid from aneuploid cells, as described in example 1, by applying the voltage and offset values in the machine template.

When applied to the CD45/SSC map, the CD45 negative aneuploid cells were tumor cells (e.g., the left green population of cells in FIG. 7);

the tumor cell population was applied to each channel, and the ratios of EGFR +, ALK +, and PD-L1+ tumor cells were obtained.

As shown in fig. 7.

The ratio of EGFR + ALK + PD-L1+ tumor cells detected by the same sample in a flow mode can obtain a cutoff value compared with the immunohistochemistry method by a conventional method, and the sample higher than the cutoff value can help a doctor patient to select a proper drug or treatment scheme by combining clinical other tests.

The method for determining the cutoff value comprises the following steps: the same sample is compared with a gold standard method by a flow method, about 20 samples are respectively detected by the flow method and the gold standard method, then data statistics is carried out, the samples with ALK indexes higher than a certain value in a flow result are judged to be positive in the gold standard, the samples with ALK indexes lower than the certain value are judged to be negative, and the value is classified as a cutoff value. The same applies to cutoff values of other indexes.

Example 3

Example 3 the same experimental procedure as in example 1 or 2 is followed, except that a selection of antibody combinations for use in the assays of the invention is provided. As shown in FIG. 8, the results of the EGFR antibody titer test indicate that the EGFR antibody can be used in an amount of 0.5ul to 2ul, and that the results of the test are better. FIG. 9 shows the antibody titer test of PD-L1, which indicates that the PD-L1 antibody can be used in an amount of 0.5ul to 2ul, and the better test results can be obtained. As shown in FIG. 10, the antibody combination test results show that the antibody combination provided by the present invention has significant peak of yin and yang, and can well group cells.

Example 4

Example 4 the same as the experimental method of examples 1-3, the control cell line was screened by flow cytometry, and the results are shown in fig. 11, three kinds of lung cancer epithelial cells, i.e., a549 cell, H1299 cell and H2228 cell, were selected by reference literature, and the H2228 cell was found to express two proteins, i.e., EGFR and ALK, simultaneously by experiment, and finally H2228 cell was selected as an EGFR/ALK positive cell.

Example 5

Detection of solid tumor markers

Control cell composition: PD-L1 positive control cell H4441, PD-L1 negative control cell Beas-2B, EGFR, ALK positive control cell H2228 and PBMC, wherein each cell is 1.0 × 10⁶Mixing the raw materials at a ratio of 1/mL to 1.

1. Fresh tumor cancer tissues stored at low temperature in RIPM (cell culture fluid) for no more than 48h are prepared into tissue suspension by a tissue suspension preparation device, and the operation is always kept on ice. 20ul of tissue suspension was taken, stained with 0.4% trypan blue and viable cells were counted.

2. Preparing single cell suspension from patient sample at least 1.0 × 10⁶Per mL;

3. taking 250 mu L of the cell suspension, 1mL of D-PBS solution and 1.5mL of a centrifuge tube, and centrifuging for 5min at room temperature of 300 g;

4. the supernatant was aspirated off;

5. adding 100-350 mu L of cell fixing solution and membrane breaking agent solution, gently blowing, resuspending the cells, and incubating for 1h at room temperature.

Antibody labeling:

1. adding 200 μ L of control cells into a 1.5mL centrifuge tube, adding 1mL of 2% BSA solution into each patient sample cell suspension and control cells, and incubating at room temperature for 5 min;

2. centrifuging for 5min at room temperature of 100-350 g;

4. antibodies were prepared according to the following system (currently available)

5. Adding 100 mu L of prepared antibody into each sample including a control sample tube, and lightly blowing, beating and uniformly mixing;

6. incubate at room temperature in the dark for 30 min.

Cell staining:

1. adding 1mL of 2-5% BSA into each incubated sample and the control tube, gently mixing, and incubating at room temperature for 5-10 min;

2. centrifuging at room temperature for 5-10min at 800 g;

4. repeating the steps for 1 time;

5. adding 100 mu L of 1 mu g/mL Cell Cycle Dye Cell staining working solution into the tube 1 and the control tube in each sample, gently blowing, beating and uniformly mixing, and incubating for 20-30min in a dark place at room temperature;

6. the analysis was performed on a machine with a PD-L1 cutoff value of 5%, an EGFR cutoff value of 5%, and an ALK cutoff value of 6%.

FIG. 12 shows the results of the live cell assay, in which the black dots outside the circled portions indicate dead cells and debris. As shown in fig. 13, tumor cell clusters can be dispersed into single cells by the methods provided by the present invention. As shown in FIG. 14, the tissue cells in the green region were CD45-, and the non-tissue cells in the blue region were CD45+, thereby differentiating the tissue cells from the non-tissue of the resulting cell suspension. Fig. 15 shows the results of the detection of euploid and aneuploid. FIG. 16 shows the results of EGFR, ALK, and PD-L1 detection. The Cutoff value is defined as that when the proportion of tumor cells expressing a certain marker in all tumor cells exceeds a certain percentage, the sample is considered as a sample positive for the marker, and the certain percentage is the Cutoff value which is obtained through calculation. Specifically, in the present example, the cutoff value of PD-L1 was calculated, and the cutoff value of PD-L1 means that the sample was determined to be positive for PD-L1 after the proportion of PD-L1-expressing tumor cells to all tumor cells exceeded 5%. Similarly, the cutoff value of the EGFR refers to that the sample is considered to be EGFR positive after the EGFR expressing tumor cells account for more than 5% of all tumor cells; the cutoff value of ALK means that when the ratio of ALK-expressing tumor cells to all tumor cells exceeds 6%, the sample is determined to be ALK positive. The cutoff value is calculated as described in example 2 and will not be described in detail here.

Example 6

Tumor microenvironment detection

Fresh tumor cancer tissues stored at low temperature in RIPM (cell culture fluid) for no more than 48h are prepared into tissue suspension by a tissue suspension preparation device, and the operation is always kept on ice.

1. The tissue suspension was centrifuged, washed once with PBS, and then stained with 0.4% trypan blue for viable cell counting

2. Preparing single cell suspension from patient sample at least 1.0 × 106/mL;

3. taking 250 mu L of the cell suspension, 1mL of D-PBS solution and 1.5mL of a centrifuge tube, and centrifuging for 5-10min at the room temperature of 300g-600 g;

4. the supernatant was aspirated off;

5. 250 μ L of cell fixative solution was added and gently pipetted, the cells were resuspended and incubated at room temperature for 1 h.

6. Adding 1mL of 2-5% BSA solution, and incubating at room temperature for 5-10 min;

7. centrifuging at room temperature for 5-10min at the temperature of 300-600 g;

8. the supernatant was aspirated away, leaving 50-100. mu.L of liquid, taking care not to aspirate the cell pellet;

9. antibodies were prepared in the following amounts (currently available)

10. Adding 100 mu L of prepared antibody, lightly blowing, beating and uniformly mixing;

11. incubating at room temperature in dark for 20-30 min.

12. Adding 1ml PBS, washing once, and putting on the machine

13. Immune cells in the microenvironment were analyzed.

FIG. 17 shows the expression status of CD4+ PD-1+, CD8+ PD1+ in the solid tumor surrounding immune cells, as shown in FIG. 17, which is the result of the analysis of the solid tumor surrounding immune cells, wherein the immune cells are analyzed without the cutoff value.

Example 7

Example 7 as with the experimental methods of examples 1-6, example 7 provides cutoff specific data as shown in table 1 (where% X45 neg refers to the proportion of 45 negative cell population).

And comparing the flow type experiment result of the same tissue sample with the IHC experiment result to obtain a cutoff value, wherein the sample higher than the cutoff value means that one or more samples have clinical significance in detecting the positive tumor marker. The positive marker of which the flow detection result is higher than the cutoff value has clinical significance, which indicates that the kit can be used for evaluating tumor treatment medicines or treatment schemes.

TABLE 1

As can be seen from the table, the total coincidence rate of EGFR and ALK was 100% and the total coincidence rate of PD-L1 was 89% in the flow assay result compared with the IHC assay result. Compared with the immunohistochemical method, the streaming method provided by the application has the advantage that the same experimental detection result is consistent. The high coincidence rate of 100% and 89% indicates that the flow detection method provided by the invention can replace the traditional immunohistochemistry and obtain accurate detection results.

Claims

1. A detection method of a solid tumor targeted index comprises the detection of a solid tumor targeted therapeutic molecular marker and the detection of a solid tumor microenvironment, and the evaluation method comprises the following steps:

step three: establishing a flow cytometer on-machine model;

step five: detecting a solid tumor microenvironment by flow cytometry;

2. The detection method of claim 1, wherein in step four, the set of molecules of the molecular markers for the solid tumor targeted therapy comprises: CD45, PD-L1, EGFR, ALK, Cell Cycle Dye.

3. The assay of claim 1 wherein in step five, solid tumor-surrounding immune cells are assayed to detect the solid tumor microenvironment, and the combination of molecules that detect solid tumor-surrounding immune cells comprises CD3, CD4, CD8, CD45, PD-1.

4. The method of claim 1, wherein in step two, the tumor tissue sample is dispersed into single cells by vortex centrifugal force.

5. The method of claim 4, wherein the tumor tissue sample has a volume of 10-20mm²The tissue mass of (1).

6. The detection method according to claim 1, wherein in step three, PD-L1 positive cells, PD-L1 negative cells, EGFR/ALK positive cells and peripheral blood mononuclear cells PBMC are mixed in a ratio of 1:1:1:1 to prepare control system cells; respectively adding cells of a control system into single-tube control tubes, wherein the single-tube control tubes are respectively named as control, EGFR, ALK, CD45, PD-L1 and cell cycle; wherein, the control tube is a negative control and does not add an incubation antibody, and the EGFR tube, the ALK tube, the CD45 tube, the PD-L1 tube and the cell cycle are used for antibody incubation; establishing an upper computer template, and performing compensation calculation on negative control non-incubated antibody io control cells; using an FSC-A graph and an SSC-A graph to machine an unincubated antibody Control tube, and adjusting the voltage to ensure that the cells are in the middle of the FSC-A graph and the SSC-A graph and the cells of each channel are in a negative position; and (3) operating the control tube and the mixing tube of each single tube, selecting cells on FSC-A and SSC-A graphs, adjusting the voltage of each channel to ensure that the negative peak and the positive peak of each channel are obviously grouped, calculating the compensation value of each fluorescence channel, and operating the computer according to the calculated compensation value.

7. The assay of claim 6, wherein preparing cells of a control cell system comprises: PD-L1 positive cells H441, PD-L1 negative epithelial cells, EGFR/ALK positive cells H2228 and peripheral blood mononuclear cells PBMC.

8. The detection method according to claim 7, wherein live cells are selected on the FSC-a/SSC-a map by loading on a voltage and compensation values in a loading template, the adhesions are removed in a cell cycle map, and the separated adhesions are applied to the cell cycle map to distinguish diploid cells from aneuploid cells; when the aneuploid cells were applied in a CD45/SSC map, the CD45 negative aneuploid cells were tumor cells.

9. The detection method of any one of claims 1 to 7, wherein the tumor cell population is applied to each channel, the ratio of tumor cells that give EGFR +, ALK +, and PD-L1+ gives a cutoff value, and samples above the cutoff value are used for the evaluation of a tumor treatment drug or treatment protocol.

10. A kit for detecting a target marker of a solid tumor, which is used in the detection method according to any one of claims 1 to 9, wherein the kit comprises a combination of fluorescently-labeled antibodies as shown below: a combination of fluorescence labeled antibodies consisting of CD45-PC7, PD-L1-APC, EGFR-FITC, ALK-PE and Cell CycleDye-BV421 or a combination of fluorescence labeled antibodies consisting of EGFR-AF488 ALK-PE, CD45-PC7, AF647-PD-L1 and Cell CycleDye; and a combination of fluorescently labeled antibodies consisting of CD45-percp, CD3-APC, CD4-PC7, CD8-AC7, and PD-1-FITC.