KR20210017294A - Methods of providing information about the level of expression of a protein, gene, or combination thereof in an individual by performing multiple marker immunohistochemical staining and/or in situ hybridizations - Google Patents

Abstract

The present invention relates to a method for using multiple-marker immunohistochemical staining and tissue microarray to provide information about an expression level of proteins, genes, or combinations thereof of an entity. A conventional method can check expression of only one gene or protein, so even if a new tissue slide is made from the same paraffin block, it was impossible to simultaneously acquire expression information of various genes and proteins in the same cell as the stained cell. However, according to one aspect of the present invention, since microarray is used, various tissues can be rearranged in a single paraffin block to implement efficient tissue management and large-scale data acquisition, and gene and protein expression information can be simultaneously acquired at a cell level through sequential in-situ matching and immunochemical staining to be usefully used in large-scale medical research using a patient′s tissues. In addition, according to one aspect of the present invention, various diagnostic/therapeutic markers are identified with a limited tissue or correlation between the markers are managed at a cell level, thereby helping treatment customized to a patient. In particular, the expression of cells, which are difficult to be identified with only one immunostaining, such as intratumoral inflammatory cells (CD39, CD73, or CD103(+) inflammatory cells, M1 macrophage, or bone marrow-derived immunosuppressive cells), can be checked through correlation with gene changes and the method can be usefully used in predicting the reactivity of a patient to an immunotherapeutic agent with minimal tissue use. The method comprises the following steps of: acquiring a sample; performing a tissue microarray; performing staining; and performing analysis.

Description

Methods of providing information about the level of expression of a protein, gene, or combination thereof by performing multi-marker immunochemical staining and/or in situ matching combination thereof in an individual by performing multiple marker immunohistochemical staining and/or in situ hybridizations}

It relates to a method of providing information on the expression level of a protein, gene, or combination thereof in an individual using multiple marker immunochemical staining and tissue microarrays.

As one of the treatment methods for treating various cancers, targeted treatments and immunotherapy are in the spotlight. However, efforts to find targeted treatments and immunotherapy products tailored to individuals are continuing, and since various markers exist accordingly, diagnosis by biopsy is increasing in order to confirm this. Therefore, there is a need to satisfy the evaluation of various markers using only the minimum amount of tissue.

In such a situation, a technology that rearranges several paraffin blocks containing tissues into a single paraffin block and simultaneously checks the expression of several genes (DNA/RNA) and several proteins on a single slide made from this block at the cell level. It is known to be suitable for use in large-scale medical research or in pathological/diagnostic clinical settings to identify patient diagnosis/treatment markers.

However, the conventional in situ matching method or immunohistochemical staining only confirmed the expression of one kind of gene or one kind of protein when applied to a single slide made from a single sample block, and different DNA/RNA or protein in the same cell. In order to see the expression of, it is necessary to make a series of slides and use a limited method of in situ conformation and immunochemical staining at similar locations. In the case of the recently marketed multiple immunofluorescence staining, several proteins were simultaneously labeled, but it was impossible to observe the overall structure of the tissue, and interference between colors was a problem, and available antibodies were limited, and still paraffin block cells. There is a lack of means to simultaneously confirm the expression of various proteins and genes within. The flow cytometry method can simultaneously check the expression of various proteins in a specific cell, but since it must be made into a single cell state, the shape of the cell cannot be known, the histological location cannot be accurately specified, and the fixed state in a paraffin block Cells cannot be analyzed. Single-cell RNA-seq is difficult to determine the relationship with proteins, and it is known that histological information is damaged during the experiment.

In addition, in order to obtain representative indicators for assessing the effectiveness of immunotherapy drugs, objectivity and reliability were problems with the existing methods, and there was a need to shorten the time required for diagnosis.

Accordingly, there is a need for an evaluation method that uses minimal tissue and evaluates the expression of various markers in a large amount at the same time, while objectivity and reliability are recognized, and the time to diagnosis is short.

One aspect is to provide a method of providing information about the expression level of a protein, gene, or combination thereof in an individual.

Another aspect is to provide a method of providing information for personalized cancer treatment.

One aspect provides a method of providing information about the expression level of a protein, gene, or combination thereof in an individual.

It will be described in more detail below.

The method comprises obtaining a biological sample of the subject; Performing a tissue microarray comprising the step of preparing a tissue slide by impregnating the obtained sample with paraffin wax; Immunochemical staining on microarray slides of the tissue; And DNA or RNA in situ hybridization; performing at least one selected from the group consisting of; And obtaining an image of the stained tissue slide and analyzing it.

The biological sample of the individual is, for example, induced pluripotent stem cells, primary cells, cell lines derived from cancerous tissues, neuronal cells, skin cells, liver cells, It may include a tissue comprising one or more selected from the group consisting of tumor cells, stromal cells, immune cells, epithelial cells, and endothelial cells, and specifically, the biological sample is, for example, a tissue including cancer cells, a cancer parenchyma (parenchym), intratumoral tissue, or peritumoral stroma.

The method includes blood, tissue, urine, mucus, saliva, tears, plasma, serum, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, serous fluid, genitourinary fluid, breast milk, lymphatic fluid, semen, cerebrospinal fluid , Pluripotent stem cells, primary cells, cell lines derived from cancerous tissues, neuronal cells derived from internal body fluid, ascites, cystic tumor body fluid, amniotic fluid, or a combination thereof ), obtaining a tissue sample derived from one or more of skin cells, liver cells, tumor cells, stromal cells, immune cells, epithelial cells, and endothelial cells. The step of obtaining a biological sample from the individual may be performed by a conventional collection and separation method.

The individual may be a mammal including a human. The individual may mean, for example, a target for predicting whether sensitivity to a target therapy or an immunotherapy appears among patients with cancer disease. The term immunotherapeutic agent has the same meaning as an immune checkpoint inhibitor, and in the present specification, an immunotherapy agent and an immune checkpoint inhibitor may be used interchangeably.

In addition, by the above method, it may be possible to confirm the expression levels of various genes and/or proteins in parenchymal tissue cells of cancer, tissue cells in the dark parenchyma, and peripheral stromal tissue cells. In addition, the step of analyzing in the method includes determining a threshold or a criterion for positive, and determining the expression pattern of various genes and/or proteins in the measured tissue as positive or negative based on this. Can include.

The individual may include a vertebrate, a mammal, or a human (Homo sapiens). For example, the human may be Asian or Korean.

In the above method, the step of preparing a tissue slide may include processing a biological sample obtained from an individual into a circle having a diameter of, for example, 0.5 mm to 2.5 mm, and attaching it to the slide. The slide may be a rearranged of the processed biological sample, for example, if the sample has a diameter of 0.6 mm, for example, 500 to 600 may be rearranged, or if the diameter is 2.0 mm, for example For example, up to 50 to 60 slides can be rearranged.

In addition, in the step of performing staining in the above method, the immunochemical staining may be a multi-marker immunochemical staining, through which expression information of various genes and proteins that are variously expressed in the tissue can be obtained simultaneously.

The expression information can be obtained by introducing a label that generates a detectable signal to an antibody, and the label that generates the detectable signal is, for example, a chemical substance (eg, biotin), an enzyme (alkaline phosphatase). , β-galactosidase, horseradish peroxidase and cytochrome P450), but not limited thereto.

Enzymes bound to the antibody used in the method include, but are not limited to, enzymes that catalyze the color development reaction, and include, for example, alkaline phosphatase, β-galactosidase, horseradish peroxidase, and lucifer. Raase and Cytochrome P450. When alkaline phosphatase is used as the enzyme that binds to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate -B1-phosphate) and ECF (enhanced chemifluorescence) are used, and when horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis -N-methylacridinium nitrate), resorupine benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine- HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD), naphthol/pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS Substrates such as (phenzaine methosulfate) may be used.

In the above method, measurement of the final expression or measurement of a signal may be carried out according to various methods known in the art. In addition, the method of aligning and analyzing the images obtained in the above method can be automated.

Another aspect is to provide a method of providing information for personalized cancer treatment.

The method includes: performing a tissue microarray including obtaining a biological sample of an individual and impregnating the obtained sample with paraffin wax to prepare a tissue slide; Multiple marker immunochemical staining on tissue slides; And DNA or RNA in situ hybridisation; performing at least one staining selected from the group consisting of; And obtaining a stained tissue slide image, measuring the level of at least one selected from the group consisting of expressed DNA, RNA, and protein, and analyzing it.

The analyzing step may determine whether the individual or the subject is a patient with high sensitivity to a target therapy or an immunotherapy based on the expression level in the expressed DNA, RNA and protein. Therefore, personalized cancer treatment may mean that an individual determines as a therapeutic target of an immunotherapy.

In addition, in the method, in the step of analyzing, for example, a combined positive score (CPS) for cancer calculated by measuring the expression level of the individual's expressed DNA, RNA or protein is greater than 1. In this case, it may include determining that the individual is highly sensitive to the immunotherapy. CPS, which is a positive score for cancer, is a representative indicator for assessing the effectiveness of an immunotherapy agent. When the indicator has a value of 1 or more, it is determined that the therapeutic effect appears, and is known to be calculated by the following formula.

In addition, in the above method, the expression of at least one protein selected from the group of surface-expressing proteins directly related to the suppression of immune cells consisting of PD-1, TIM, and LAG3 is measured, and the expression of the protein is responded to the drug. If the expression level is higher than the average level of cancer patients estimated to be absent, it may include determining that the individual is highly sensitive to the immune checkpoint inhibitor. The PD-1, TIM and LAG3 are exemplary, and even markers involved in immune checkpoint inhibition can be used without limitation.

In the above method, the biological sample may be, for example, cancer cells, cancer parenchyma, dark parenchymal tissue, or cancer surrounding stromal tissue cells. Tissue is largely divided into parenchyma and epilepsy. Interstitial tissue refers to non-unique components such as connective tissue and blood vessels accompanying the parenchyma, and the parenchyma of cancer refers to tumor cells that are the main components. The tissue in the dark parenchyma refers to a tissue including other cells, such as inflammatory cells or blood vessel cells, which are in contact with tumor cells or sandwiched between adjacent tumor cells.

In addition, the above method enables simultaneous identification of the expression levels of various genes and/or proteins in the parenchymal tissue cells of the cancer, the tissue cells in the dark parenchyma, and the surrounding stromal tissue cells, so that the parenchyma of cancer, tissue cells in the dark parenchyma or surrounding stromal tissue cells Cells that simultaneously express the immune checkpoint inhibitor can also be distinguished by a method according to an aspect.

In addition, the cancer is lung cancer, laryngeal cancer, stomach cancer, colon/rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, kidney cancer, bone cancer, muscle cancer, fat cancer, leukemia, lymphoma, hematologic cancer. , And may be one or more selected from the group consisting of a malignant tumor occurring in the nervous tissue.

According to the conventional method, only one gene or protein expression can be checked, so even if a new tissue slide is made from the same paraffin block, it is impossible to obtain the expression information of various genes and proteins in the same cell as the stained cell. According to a method according to one aspect, by rearranging various tissues in one paraffin block by using a microarray, it is possible to efficiently manage tissues and obtain large-scale data. Since protein expression information can be obtained at the same time in cell units, it can be used effectively in large-scale medical research using patient tissues. In addition, according to an aspect of the method, it is possible to assist in customized treatment of patients by identifying various diagnostic/treatment markers with limited tissues or managing correlations between markers at the cellular level. In particular, the expression of cells that are difficult to identify with only one immunostaining, such as inflammatory cells in tumors (CD39, CD73, or CD103(+) inflammatory cells, M1 macrophages, or bone marrow-derived immunosuppressive cells, is confirmed to be associated with changes in genes. In addition, it can be usefully used in predicting the responsiveness of patients to immunotherapy with minimal tissue use.

1 is a diagram schematically illustrating an image analysis process through expression of genes including proteins and DNA and RNA using tissue microarray slides.
2 is a diagram illustrating the expression patterns of tissue proteins and RNA of the prepared tissue microarray slide.
3 is a diagram showing the results obtained by performing expression analysis on CD8 and CD3 positive inflammatory cells according to an aspect of the method.
4 is a diagram showing that each slide is aligned with the same coordinates in order to confirm the expression patterns of various markers.

Hereinafter, an aspect will be described in more detail through examples and experimental examples. However, these Examples and Experimental Examples are for illustrative purposes only, and the scope of one aspect is not limited to these Examples and Experimental Examples, and Examples and Experimental Examples of an aspect are average knowledge in the art. It is provided to explain one aspect more completely to those who have.

Experimental Example 1. Fabrication of tissue microarray (TMA)

Tissue slides made from paraffin blocks were checked with an optical microscope, and the area of interest was marked in a circle. After finding the position of the same paraffin block as the area on the labeled tissue slide, the tissue (core) was collected in a circular shape having a diameter of 0.6 mm or 2.0 mm using a trephine apparatus. The harvested tissue is rearranged on a new paraffin block. When the diameter was 0.6 mm, 556 pieces were placed in one new paraffin block, and when the diameter was 2.0 mm, up to 60 tissues were placed in one new paraffin block. Subsequently, from the rearranged paraffin block, all the cores were sliced to be included at the same time and attached to a glass slide to prepare a tissue microarray.

Experimental Example 2. Performing multiple marker immunochemical staining and RNA in situ hybridization

In the multi-marker immunochemical staining method and RNA in situ hybridization method, tissue immunochemical staining and in situ hybridization were performed in the following order to identify DNA, RNA, or protein. The prepared tissue microarray was deparaffinized and hydrated. Each solution used at this time was dispensed into a separate glass container and used. The specific process is as follows. The prepared glass slide was immersed in xylene I, II, and III for 5 minutes each. Then, the slides were immersed in 100% alcohol I and II for 5 minutes each, 95%, 80%, and 70% alcohol were each immersed for 1 minute, and finally, the slides were immersed in tap water for 10 minutes.

In addition, the attached antibody was recovered through the following procedure. The target antibody recovery solution and antibody recovery method were determined according to the manual by referring to the data sheet of the manufacturer of the primary antibody. After that, the slides were allowed to stand at room temperature for 20 minutes to dry, and washed three times for 5 minutes with tap water.

Thereafter, the slide was blocked in methanol methanol and 0.3% H ₂ O ₂ for 10 minutes for the purpose of blocking endogenous peroxidase, and the slide was tris-buffered saline (TBS) and Tween containing 4% skim milk. Blocking non-specific proteins with 20 (TBS-T). Then, it was washed three times in tap water for 5 minutes.

After dispensing the primary antibody onto the slide, it was covered with a paraffin film to prevent evaporation, and incubated in a humid chamber for 2 hours at room temperature or at 4°C overnight. Thereafter, the primary antibody was cultured by washing twice with distilled water containing 0.2% Tween 20. Subsequently, the secondary antibody culture was amplified by dispensing an unconjugated secondary antibody to the animal from which the primary antibody was generated to the slide, and incubated at room temperature for 45 minutes to 1 hour. Then, it was carried out by washing twice with distilled water containing 0.2% Tween-20.

Horseradish peroxidase (HRP) binding antibody Dako REAL EnVision HRP RABBIT/MOUSE (K5007, Dako) was sufficiently dispensed on the slide and incubated at room temperature for 1 hour with a humidifier. Then, it was washed three times in tap water for 5 minutes. Thereafter, in order to confirm the color development, color development was performed using ImmPACT AEC (SK-4805, VECTOR), and the staining result of the tissue sample was checked, and then washed with tap water for 10 minutes. For counterstaining and mounting, hematoxylin was diluted 1:9, stained for 45 seconds, and washed with tap water for 10 minutes. Thereafter, a coverslip was enclosed on the slide using a Clearmount mounting solution (Cat no.008010, Thermo Fisher). The RNA/DNA in situ hybridization was performed according to the protocol for each product, and when Roche's EREB1 in situ hybridization was performed, the process is as follows. After digesting the slides with Proteinase K, a dinitrophenyl (DNP)-binding probe targeting EREB1 viral RNA was hybridized at 37°C for 2 hours. After amplifying the signal with a rabbit anti-DNP antibody and a goat anti-rabbit secondary antibody conjugated with HRP, confirm that the RNA is marked with black dots using metallic silver ions, and perform the in-situ matching method. Performed.

Experimental Example 3. Checking the stained slide image using a slide scanner

The obtained slide image was scanned at 400x magnification using an Aperio Scanscope slide scanner, and at least one focus was set for each core, and the creation of an svs format image file was confirmed.

Experimental Example 4. Initialization of stained slides

4.1 Initialization of stained protein slides

The coverslip was separated from the slide, and the final 300 ml of 20% sodium dodecyl sulfate (SDS) 30 ml, tris HCl buffer 0.5 M solution (pH 6.8) 37.5 ml, 2-mercapto ethanol 2.4 ml and distilled water 232 ml were mixed A stripping solution was prepared. After that, the slide was shaken for 30 minutes at 55°C in a stripping solution. Subsequently, the initialized slides were subjected to the immunostaining or in-situ method of Experimental Example 3.

4.2 Initialization of stained gene slides

After separating the coverslip from the slide, treating the stripping solution according to the kind and color development of the gene to be identified, or waiting for natural decolorization, the initialized slide was subjected to the in situ conformation method or immunostaining of Experimental Example 3.

Experimental Example 5. Array extraction/alignment and automated analysis of tissue microarray images

Using QuPath, the tissue microarrays in the svs file were captured by staining/core, each coordinate was obtained, grips were set with Aperio ImageScope, and images were cut out in tif file format for each core and saved. Thereafter, the crop core image was separated by UnMixColors using the Cellprofiler, and then the hematoxylin color was separated and aligned based on this to calculate the coordinate error (difference between the x and y axes). Using the error data obtained from Cellprofiler, cores per dye were obtained at the same location without compromising resolution. Accurate core alignment was performed using a Cellprofiler, and AEC stained with unMixColors was colored differently according to antibodies, and then multiplexed analysis was performed. Observe the overlapping areas between staining using the MaskObject of Cellprofiler, get information on the number, size, shape, and degree of staining of cells using the Identifiy Primary Object, and convert the contents into pictures using Convert Object To Image 　SaveImages function The image was saved and checked. The subsequent process was automated through the AutoHotkey script, and the overall image analysis process is schematically shown in FIG. 1.

Example 1. Cell-specific gene and protein expression and density confirmation

For each of the prepared TMA cores, a positive threshold or a reference point was obtained for each gene and protein, and the results of confirming the tissue expression pattern by dividing it into positive and negative, respectively, are shown in FIG. 2. As confirmed in FIG. 2, cytokeratin, a protein that proves that it is an epithelial cell in the epithelial cells of the endometrium, is a single slide with tissue immunochemical staining shown on the left side, and ERER1, a confirmed method for the Epstein-Barr virus, shown on the right side. As confirmed by the in-situ matching method, it was confirmed that the part marked with a circle in the displayed image was positive, and it was confirmed that it was the same location in the tissue, and it was confirmed that it can be effectively represented as one image.

Analysis of CD8 and CD3 positive inflammatory cells was performed using Matlab, which is shown in FIG. 3. As confirmed in FIG. 3, it was confirmed that most of the CD8-positive inflammatory cells were expressed together with the CD3-positive inflammatory cells, and it was confirmed that antibodies to CD8 were expressed in a CD3 T cell-specific subtype.

Accordingly, it is possible to comprehensively divide the expression of tissue genes and proteins into either positive or negative, and it was possible to effectively identify genes and proteins expressed for each cell in addition to one slide.

Example 2. Target therapy/immunotherapy application target complex evaluation

An experiment was conducted to confirm whether an individual could be selected as a target for administration of a specific target therapy or immunotherapy by confirming the expression of various markers from a biological sample of a patient using a minimal amount of tissue. Since the multi-tissue microarray in situ method and immunohistochemical staining method can use only a small number of tissues, according to the experimental examples described above, ERBB2 was used to determine whether an individual is highly sensitive to the target treatment, Trastuzumab. It was confirmed by the method through gold standard DNA in situ conformance staining. In addition, gold standard RNA in situ matching tissue staining for EBER1 and EBV was performed to determine whether an individual is highly sensitive to anti-PD1 therapy, and PD to determine whether the individual is highly sensitive to various immunosuppressants. Protein in situ conformance staining was performed for -L1, and the in situ conformance method and immunohistochemical staining were successively performed and aligned with the same coordinates as shown in FIG. 4. As a result of performing tissue staining from biological samples obtained from 59 gastric cancer patients and evaluating it, ERBB2 was positive in 7 patients (11.8%) in one tissue microarray, and EBER2 was positive in 3 patients (5.0%). It was confirmed that it was positive. Overall, it was confirmed that it is possible to effectively confirm that 7 patients with high sensitivity to trastuzumab and 3 patients with high sensitivity to immunosuppressants.

Example 3. Evaluation for determining the sensitivity of a specimen to an immunotherapeutic agent

Currently, a combined positive score (CPS) in gastric cancer exists as a representative indicator for assessing the effectiveness of immunotherapy drugs, which is judged to have a therapeutic effect when it has a value of 1 or more. It is known to calculate.

To check whether the above value can be obtained accurately while improving objectivity and reliability through the results shown on one slide through the multi-tissue microarray in situ method and immunohistochemical staining method described above, and shortening the time required for diagnosis. The experiment was carried out.

The number of inflammatory cells and PD-L1 were positive by staining for the number of cancer cells and the CD45 marker through cytokeratin staining in gastric cancer samples collected from 59 patients using 2000x2000 pixels in the center of one microstructure array. The cells indicated by were counted through each slide, and the results of obtaining CPS values are shown in Table 1. The equation used to find the CPS is as follows. CPS=((1)+(2))/(3)X100

Sample number PD-L1 in cancer cells(1) PD-L1 in immune cells(2) # of cancer cells(3) CPS Treatment Core1 0 3 229 1.31 One Core2 0 2 334 0.60 0 Core3 One 0 703 0.14 0 Core4 0 11 0 NA One Core5 0 8 308 2.60 One Core6 0 11 149 7.38 One Core7 0 One 235 0.43 0 Core8 0 4 328 1.22 One Core9 0 One 197 0.51 0 Core10 0 0 25 0.00 0 Core11 0 One 45 2.22 One Core12 0 3 72 4.17 One Core13 2 12 160 8.75 One Core14 0 17 41 41.46 One Core15 0 24 134 17.91 One Core16 13 5 178 10.11 One Core17 0 0 925 0.00 0 Core18 0 0 233 0.00 0 Core19 0 5 165 3.03 One Core20 0 7 100 7.00 One Core21 0 0 1083 0.00 0 Core22 0 2 384 0.52 0 Core23 One 13 294 4.76 One Core24 35 34 261 26.44 One Core25 0 3 0 NA One Core26 0 6 245 2.45 One Core27 2 58 120 50.00 One Core28 0 5 94 5.32 One Core29 0 0 34 0.00 0 Core30 0 12 663 1.81 One Core31 0 One 198 0.51 0 Core32 0 One 50 2.00 One Core33 0 15 120 12.50 One Core34 0 3 129 2.33 One Core35 0 12 657 1.83 One Core36 0 2 42 4.76 One Core37 0 3 0 NA One Core38 2 14 386 4.15 One Core39 0 13 3 100.00 One Core40 0 0 511 0.00 0 Core41 One 0 145 0.69 0 Core42 0 One 52 1.92 One Core43 0 One 90 1.11 One Core44 0 One 115 0.87 0 Core45 0 6 122 4.92 One Core46 0 0 263 0.00 0 Core47 0 4 161 2.48 One Core48 One 6 132 5.30 One Core49 0 2 73 2.74 One Core50 0 0 70 0.00 0 Core51 0 6 22 27.27 One Core52 0 One 470 0.21 0 Core53 0 3 508 0.59 0 Core54 0 One 10 10.00 One Core55 One 9 385 2.60 One Core56 0 0 330 0.00 0 Core57 0 2 21 9.52 One Core58 4 12 703 2.28 One Core59 One 0 383 0.26 0

*NA: CPS evaluation impossible due to the absence of cancer cells in the center of the core

As confirmed in Table 1 above, when CPS was evaluated by the multi-tissue microarray in situ method and immunohistochemical staining method, it was confirmed that it had a value of 1 or more in a total of 39 samples. Therefore, it can be judged with high objectivity and reliability that it is possible to easily determine an individual corresponding to a sample with high sensitivity to immunotherapy among 59 samples with one slide through the multi-tissue microarray in situ method and immunohistochemical staining method. It was confirmed that it can be.

Example 4. Expression analysis of immune checkpoint inhibitors according to subtypes of immune cells

The expression of PD-1, TIM, and LAG3, known as immune checkpoint inhibitors expressed in T lymphocytes, in the intratumoral and peritumoral stromas of T lymphocytes is determined by the indirect T cell subtype. An experiment was conducted to see if it changed. Since the parenchyma of cancer and the surrounding epilepsy can be distinguished by the expression of cytokeratin protein, the cytokeratin protein was used to differentiate between cancer cells and surrounding stromal cells, and the expression of immune checkpoint inhibitors expressed in T cells was confirmed. It is shown in Table 2.

Inhibitor All T cells Cytotoxic T cells Regulator T cell Helper T cell PD-1 CD45+/CD3+/PD-1+ CD45+/CD3+/CD8+/PD-1+ CD45+/CD3+/CD8-/FOXP3+/PD-1+ CD45+/CD3+/CD8-/FOXP3-/PD-1+ TIM CD45+/CD3+/TIM+ CD45+/CD3+/CD8+/TIM+ CD45+/CD3+/CD8-/FOXP3+/TIM+ CD45+/CD3+/CD8-/FOXP3-/TIM+ LAG3 CD45+/CD3+/LAG3+ CD45+/CD3+/CD8+/LAG3+ CD45+/CD3+/CD8-/FOXP3+/LAG3+ CD45+/CD3+/CD8-/FOXP3-/LAG3+

From the results confirmed in Table 2, it was possible to confirm which immune checkpoint inhibitor was expressed in which subtype of T lymphocyte by using the following protein combinations of each T lymphocyte. In addition, through the above results, it was possible to confirm cells expressing two or more inhibitors at the same time, and it was specifically confirmed that it can be confirmed through one slide. Through the above results, by identifying the marker expressed in T lymphocytes, the type of the inhibitor of the expressed T cells can also be known, so the possibility of customizing anticancer therapy by administering the appropriate anti-immune inhibitor checkpoint inhibitor. Was confirmed.

Claims (10)

As a method of providing information on the expression level of a protein, gene or a combination thereof of an individual comprising the following steps,
Obtaining a biological sample of the subject;
Performing a tissue microarray comprising the step of preparing a tissue slide by impregnating the obtained sample with paraffin wax;
Immunochemical staining on the tissue microarray slides; And DNA or RNA in situ hybridization; performing one or more staining selected from the group consisting of; And
A method comprising the step of obtaining an image of the stained tissue microarray slide and analyzing it. The method according to claim 1, wherein the biological sample of the individual is induced pluripotent stem cells, primary cells, cell lines derived from cancerous tissues, neuronal cells, skin cells, liver cells. , A tumor cell, a stromal cell, an immune cell, an epithelial cell, and an endothelial cell. The method of claim 1, wherein the step of preparing the tissue microarray slide comprises processing the tissue into a circle having a diameter of 0.5 mm to 2.5 mm and attaching the tissue to the slide. The method of claim 1, wherein in the step of performing the staining, the immunochemical staining is a multi-marker immunochemical staining. As a method of providing information for personalized cancer treatment comprising the following steps,
Performing a tissue microarray comprising the step of obtaining a biological sample of the subject and impregnating the obtained sample with paraffin wax to prepare a tissue slide;
Multiple marker immunochemical staining on tissue microarray slides; And DNA or RNA in situ hybridization; performing one or more staining selected from the group consisting of; And
A method comprising the step of obtaining a stained tissue microarray slide image, measuring the level of at least one selected from the group consisting of expressed DNA, RNA, and protein, and analyzing the same. The method of claim 5, wherein the personalized cancer treatment is determined by the individual as a target treatment and an immunotherapy treatment target. The method of claim 5, wherein the analyzing comprises immunizing the individual when the combined positive score (CPS) for cancer calculated by measuring the expression level of the expressed DNA, RNA or protein exceeds 1 The method comprising the step of determining that the sensitivity to the therapeutic agent is high. The method according to claim 5, measuring the expression of one or more proteins selected from the group consisting of PD-1, TIM, and LAG3, and determining that the individual is highly sensitive to the immune checkpoint inhibitor when the protein is high. The method comprising a. The method of claim 5, wherein the biological sample is a cancer parenchym, an intratumoral tissue, or a peritumoral stroma. The method of claim 5, wherein the cancer is lung cancer, laryngeal cancer, gastric cancer, colon/rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, kidney cancer, bone cancer, muscle cancer, fat cancer, leukemia, The method of one or more selected from the group consisting of lymphoma, hematologic cancer, and malignant tumors occurring in nervous tissue.

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