CN111235273A - Colorectal cancer tumor microenvironment detection reagent, kit, device and application - Google Patents
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Abstract
The invention provides a colorectal cancer tumor microenvironment detection reagent, a kit, a device and application. The detection reagent comprises a POLE gene mutation detection reagent and an antibody of at least one molecular marker in at least one combination of the following reagents: combination 1: CD3, CD8, CD45RO, PD-1 and PD-L1; and (3) combination 2: CD4, FOXP3, CD68, CD163, PD-L1 and CD 57. The application finds that the POLE gene mutation is obviously related to the high expression of the partial molecular markers, and the relationship between the POLE gene mutation and the colorectal cancer tumor microenvironment is determined, so that the multiple immunohistochemical methods are conveniently and comprehensively utilized to simultaneously mark a plurality of molecular markers, the detection sensitivity is high in the aspect of detecting the tumor microenvironment of a pancreatic patient, the advantage of good specificity is realized, and the 5-year survival rate of a detected object with higher probability is relatively accurately predicted.
Description
Technical Field
The invention relates to the field of multiple immunohistochemical detection, in particular to a colorectal cancer tumor microenvironment detection reagent, a kit, a device and application.
Background
The principle behind the color development of conventional immunohistochemistry with diaminobenzidine (i.e., 3, -diaminobenzidine, DAB) is that diaminobenzidine is a chromogenic substrate for peroxidase and exhibits accumulation of color change upon electron loss in the presence of hydrogen peroxide, forming a tan insoluble product. DAB is mainly combined with NH2 or SH groups of proteins to form stable N-N bonds or N-S bonds, and then chromogenic groups in DAB can display colors and mark the exposed proteins so as to display the protein distribution, the protein types and the like of target cells. The prior art typically labels a molecule on a slide.
Multiplex immunohistochemistry (mhhc) analysis: multiple immune cells and immune checkpoint molecules can be labeled on a tissue section (n is less than or equal to 6, and n represents the number of molecular markers or antigen markers).
First molecular (antigen) labeling: similar to the basic principle of conventional immunohistochemistry, primary antibody recognizes antigen, secondary antibody with Horseradish Peroxidase (HRP) binds to the primary antibody, and tyramine salt (Tyramide) with a fluorescent group forms an enzymatic product containing a covalent bond binding site under the catalysis of Horseradish Peroxidase (HRP) in a reaction containing H2O2, and binds to surrounding protein residues (including tryptophan, histidine and tyrosine residues) including on the antigen, on the primary antibody and on the secondary antibody, so that a large amount of Tyramide-fluorescent group exists at the antigen-antibody binding site, and the more the antigen, the more Tyramide-fluorescent group, the stronger the detection signal.
Nth molecular (antigen) labeling: after each antigen labeling cycle is finished, the primary antibody and the secondary antibody of the previous round are removed by using a microwave method (because the antigen-antibody reaction is the non-covalent bond combination of the molecular surface, the formed compound is not firm and can be dissociated at any time, the microwave method has little influence on the fluorescence signal of tyramine salt labeled on the antigen, so that the antibody of the previous round can be completely removed on the premise of ensuring that the labeling signal of the previous round is not lost, and the interference on the labeling of the next round is avoided. And eluting to remove the non-specific markers, and performing the next round of marking, thereby realizing the multiple immunohistochemical analysis.
The advantages are that: multiple molecules may be labeled on the same slide. In the process of labeling multiple molecules, tyramine salts with fluorescent groups of different wavelengths can be covalently bound to target antigens (directly labeling the antigens) with different primary antibodies from the same species in a sequential single-label manner, and then the antibodies are removed by microwave heating (the antibodies are not present but the fluorescent signals remain on the antigens). After the last round of antibody is cleared, new antibody can be used for continuous staining without worrying about cross reaction.
After multiple immunohistochemical labeling, multispectral imaging needs a specific analysis instrument to achieve the purpose of multiple immunohistochemical analysis. Multispectral imaging relies on both spectral data acquisition and spectral splitting calculations. Collecting spectral data: there are many technical means for multispectral data collection, such as grating spectroscopy, prism spectroscopy, liquid crystal tunable filter spectroscopy, etc. The spectral signals of specific wave bands are collected by filtering with a Vectra system (liquid crystal tunable filter, LCTF) of Perkinelmer company. The LCTF is made of liquid crystal material, changes the optical path of light in the crystal by adjusting additional voltage, selectively outputs optical signals with specific wavelength, and achieves the purpose of light splitting. The CCD exposure is matched with the continuous filtering of the LCTF, so that the image signals of different wavelength bands can be accurately recorded.
Spectrum splitting: each pixel point signal of the spectral image is the superposition of different dyes and a sample spontaneous signal, the spectral characteristic curve of each dye is taken as a standard, and reduction operation is carried out on the superposed signals in the spectral image by a mathematical method, so that the process of obtaining a single-channel image is called spectral splitting calculation. The spectrum splitting calculation is an indispensable important link of the whole spectrum imaging, and the accuracy of a data result is directly influenced.
The 'pure spectrum splitting algorithm' can be used for splitting color signals with up to 10 superposed colors, and the 'pure' dye signals hidden in the spectrum image are accurately resolved to obtain the specific distribution of each dye in the image. And the real target signal can be extracted from the autofluorescence background to obtain an image with ultrahigh signal-to-noise ratio, so that the weakly expressed fluorescence signal can be shown from the background.
Therefore, the spectrum collection and splitting method can provide guarantee for multiple immunohistochemical analysis. Multiple immunohistochemical analysis is an important means for pathological detection, and by taking colorectal cancer as an example, at present, a plurality of clinical research results suggest that the tumor immune microenvironment of the colorectal cancer is closely related to clinical prognosis outcome. A multicenter clinical study published in the Lancet journal discusses the relationship between tumor immune microenvironment immune cell infiltration and clinical prognosis, which included 3539 surgically treated stage I-III colorectal cancer patients, and the density of intraepithelial (CT) and marginal (IM) infiltrating CD3+ and CD8+ T cells was quantified to obtain an immune score. The results show that the lower the risk of relapse, the higher disease-free survival (DFS) and Overall Survival (OS) for patients with colorectal cancer whose immune score is high. A further clinical study published in the NEJM journal discusses the relationship between tumor immune cell infiltration (CD45RO +) and the invasion and prognosis of early tumor metastasis, which is included in 959 (1986) patients with colorectal cancer treated by II-IV surgery, wherein 327 patients received 5-fluorouracil-based adjuvant chemotherapy and the relationship between effector memory T cells (CD45RO +) and invasion of early metastasis (VELIPI), pathological stages (N and M stages) and survival rate was studied, which indicates that the number of CD45RO + cells in VELIPI-negative tumor patients is significantly higher than that in VELIPI-positive patients; high density infiltrating memory T cells are associated with lymph node involvement and metastasis (P < 0.001); late lymph node invasion (N2, N3) was associated with low density infiltration of CD45RO + cells; multifactorial analysis indicates that M, T, N and CD45RO + are independent predictors of OS; the OS and DFS rates were significantly higher in patients with high infiltration of effector memory T cells (CD45RO +) than in patients with lower infiltration of effector memory T cells (CD45RO +).
Although the above research results show the relationship between immune cells and colorectal cancer from different aspects, there is no relevant technical solution in the prior art for the overall research on the tumor immune microenvironment of colorectal cancer patients to evaluate the prognosis of patients, etc.
Disclosure of Invention
The invention mainly aims to provide a colorectal cancer tumor microenvironment detection reagent, a kit, a device and application, and aims to solve the problem that no related scheme exists in the prior art for detecting a colorectal cancer tumor microenvironment.
In order to achieve the above object, according to one aspect of the present invention, there is provided a colorectal cancer tumor microenvironment detection reagent, which comprises a poll gene mutation detection reagent and an antibody against at least one molecular marker in at least one combination of: combination 1: CD3, CD8, CD45RO, PD-1 and PD-L1; and (3) combination 2: CD4, FOXP3, CD68, CD163, PD-L1 and CD 57.
Further, the detection reagent comprises at least an antibody of the CD45RO molecular marker; preferably, the detection reagent comprises antibodies to at least two molecular markers CD8 and CD45 RO; more preferably, the detection reagent comprises antibodies to at least the following molecular markers: CD3, CD8, CD45RO, PD1, PD-L1 and CD 4.
Further, the antibody of the molecular marker is marked as a primary antibody, and the detection reagent also comprises a secondary antibody, preferably, the secondary antibody is selected from horseradish peroxidase, PV-6001, PV-6002 or PV-8000; preferably, the detection reagent also includes a luminescent reagent, more preferably, the luminescent reagent is selected from tyramine salts with fluorescent groups, 520-FITC, 540-AF517, 570-Cy3, 620-Cy3.5, 650-Cy5, or 690-Cy5.5.
According to a second aspect of the application, a colorectal cancer tumor microenvironment detection kit is provided, and the kit comprises any one of the colorectal cancer tumor microenvironment detection reagents.
According to a third aspect of the present application, there is provided a use of a poll gene mutation detection reagent and an antibody to at least one molecular marker in at least one combination of the following for the preparation of a kit related to the tumor microenvironment for the detection of colorectal cancer, combination 1: CD3, CD8, CD45RO, PD-1 and PD-L1; and (3) combination 2: CD4, FOXP3, CD68, CD163, PD-L1 and CD 57.
Further, antibodies to molecular markers include antibodies to CD45 RO; preferably an antibody comprising CD8 and an antibody comprising CD45 RO; more preferably, an antibody comprising at least the following molecular markers: CD3, CD8, CD45RO, PD1, PD-L1 and CD 4.
Further, the application includes: detecting whether the POLE gene is mutated or not by using a POLE gene mutation detection reagent; and detecting the molecular markers by adopting a multiple immunohistochemistry method, preferably detecting the cell positive rate of at least two molecular markers as follows: the cell positive rate of CD45RO +, the cell positive rate of CD8+, and the cell positive rate of CD8+ CD45RO +; more preferably, the cell positivity rate of CD45RO + and at least one of the following molecular markers is detected: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, PD-L1+, and CD4 +; preferably, whether the cell positive rate of CD45RO + is greater than or equal to 4.61%, whether the cell positive rate of CD8+ is greater than or equal to 2.78%, whether the cell positive rate of CD8+ CD45RO + is greater than or equal to 0.30%, whether the cell positive rate of CD3+ is greater than or equal to 5.83%, whether the cell positive rate of CD4+ is greater than or equal to 6.42%, whether the cell positive rate of CD3+ PD1+ is greater than or equal to 0.75%, and whether the cell positive rate of PD-L1+ is greater than or equal to 2.24% is detected in the case of the POLE gene mutation.
Further, applications include statistical cellular positivity rates of intraepithelial CD45RO + and at least one of the following molecular markers: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, CD8+ PD1+, CD4+, CD4+ FOXP3+, CD68+, CD68+ CD163+, CD68+ CD163-, PDL1+, and CD57 +; preferably, whether the cell positive rate of each molecular marker is greater than or equal to the corresponding threshold value is counted, if so, the 5-year productivity of the detection sample is defined as a first probability, otherwise, the 5-year productivity of the detection sample is defined as a second probability, and the first probability is greater than the second probability; more preferably, the threshold value of the cell positive rate of CD3+ is not less than 1.313%, the threshold value of the cell positive rate of CD8+ is not less than 0.876%, the threshold value of the cell positive rate of CD45RO + is not less than 0.360%, the threshold value of the cell positive rate of CD8+ CD45RO + is not less than 0.017%, the threshold value of the cell positive rate of CD3+ PD1+ is not less than 0.246%, the threshold value of the cell positive rate of CD8+ PD1+ is not less than 0.019%, the threshold value of the cell positive rate of CD4+ is not less than 1.775%, the threshold value of the cell positive rate of CD4+ FOXP3+ is not less than 0.262%, the threshold value of the cell positive rate of CD68+ is not less than 0.355%, the threshold value of the cell positive rate of CD68+ CD 163-is not less than 0.208%, the threshold value of the cell positive rate of CD68+ CD163+ is not less than 0.005%, and.
Further, the application also comprises the step of counting a first probability of the corresponding 5-year survival rate when the cell positive rate of each molecular marker is greater than or equal to the corresponding threshold value, or counting a second probability of the corresponding 5-year survival rate when the cell positive rate of each molecular marker is less than the corresponding threshold value; preferably, when the cell positive rate of CD3+ is greater than or equal to the corresponding threshold, the first probability of corresponding 5-year survival rate is 89%, and when it is less than the corresponding threshold, the second probability of corresponding 5-year survival rate is 64%; when the cell positive rate of the CD8+ is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 100%, and when the cell positive rate is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 67%; when the cell positive rate of the CD45RO + is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 81%, and when the cell positive rate is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 51%; when the cell positive rate of the CD8+ CD45RO + is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 85%, and when the cell positive rate is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 68%; when the cell positive rate of the CD3+ PD1+ is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 100%, and when the cell positive rate is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 68%; when the cell positive rate of the CD8+ PD1+ is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 69%; when the cell positive rate of the CD4+ is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 66%; when the cell positive rate of the CD4+ FOXP3+ is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 83%, and when the cell positive rate is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 72%; when the cell positive rate of the CD68+ is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 67%; when the cell positive rate of the CD68+ CD 163-is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 93%, and when the cell positive rate is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 66%; when the cell positive rate of the CD68+ CD163+ is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 81%, and when the cell positive rate is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 66%; when the cell positive rate of PDL + is more than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 72%, and when the cell positive rate is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 81%; when the cell positive rate of CD57+ is greater than or equal to the corresponding threshold, the first probability of corresponding 5-year survival rate is 100%, and when it is less than the corresponding threshold, the second probability of corresponding 5-year survival rate is 70%.
According to a fourth aspect of the present application, there is provided a colorectal cancer tumor microenvironment detection device, which is internally provided with a colorectal cancer detection molecule, wherein the colorectal cancer detection molecule includes at least one molecular marker of a detection point gene and at least one of the following combinations: combination 1: CD3, CD8, CD45RO, PD-1 and PD-L1; and (3) combination 2: CD4, FOXP3, CD68, CD163, PD-L1 and CD 57.
Further, the device incorporates a first reference marker, and in the case where the point gene is mutated, the first reference marker includes the reference cell positivity of the molecular marker CD45RO + and at least one of the following molecular markers: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, PD-L1+, and CD4 +; preferably, the reference cell positive rate of CD45RO + is not less than 4.61%, the reference cell positive rate of CD8+ is not less than 2.78%, the reference cell positive rate of CD8+ CD45RO + is not less than 0.30%, the reference cell positive rate of CD3+ is not less than 5.83%, the reference cell positive rate of CD4+ is not less than 6.42%, the reference cell positive rate of CD3+ PD1+ is not less than 0.75%, and the reference cell positive rate of PD-L1+ is not less than 2.24%.
Furthermore, the device is also internally provided with a second reference index, and the second reference index is any one or more of the following cell positive rate reference threshold values under endothelium: a cell-positivity reference threshold of CD3+, a cell-positivity reference threshold of CD8+, a cell-positivity reference threshold of CD45RO +, a cell-positivity reference threshold of CD8+ CD45RO +, a cell-positivity reference threshold of CD3+ PD1+, a cell-positivity reference threshold of CD8+ PD1+, a cell-positivity reference threshold of CD4+, a cell-positivity reference threshold of CD4+ FOXP3+, a cell-positivity reference threshold of CD68+, a cell-positivity reference threshold of CD68+ CD163-, a cell-positivity reference threshold of CD68+ CD163+, and a cell-positivity reference threshold of PDL1 +; preferably, the reference threshold value of the cell positivity of CD3+ is not less than 1.313%, the reference threshold value of the cell positivity of CD8+ is not less than 0.876%, the reference threshold value of the cell positivity of CD45RO + is not less than 0.360%, the reference threshold value of the cell positivity of CD8+ CD45RO + is not less than 0.017%, the reference threshold value of the cell positivity of CD3+ PD1+ is not less than 0.246%, the reference threshold value of the cell positivity of CD8+ PD1+ is not less than 0.019%, the reference threshold value of the cell positivity of CD4+ is not less than 1.775%, the reference threshold value of the cell positivity of CD4+ FOXP3+ is not less than 0.262%, the reference threshold value of the cell positivity of CD68+ is not less than 0.355%, the reference threshold value of the cell positivity of CD68+ CD 163-is not less than 0.208%, the reference threshold value of the cell positivity of CD68+ CD163+ is not less than 0.005%, and the.
Furthermore, the device also comprises a third reference index and a fourth reference index, wherein the third reference index is a first reference probability of the 5-year survival rate corresponding to the condition that the cell positivity of any one or more molecular markers is greater than or equal to the cell positivity reference threshold value of the corresponding molecular marker, and the fourth reference index is a second reference probability of the 5-year survival rate corresponding to the condition that the cell positivity of any one or more molecular markers is greater than or equal to the cell positivity reference threshold value of the corresponding molecular marker: when the cell positive rate of the CD3+ is greater than or equal to the cell positive rate reference threshold of the CD3+, the first reference probability of the corresponding 5-year survival rate is 89%, and when the cell positive rate is less than the cell positive rate reference threshold of the CD3+, the second reference probability of the corresponding 5-year survival rate is 64%; when the cell positive rate of the CD8+ is greater than or equal to the cell positive rate reference threshold of the CD8+, the first reference probability of the corresponding 5-year survival rate is 100%, and when the cell positive rate is less than the cell positive rate reference threshold of the CD8+, the second reference probability of the corresponding 5-year survival rate is 67%; when the cell positive rate of the CD45RO + is greater than or equal to the cell positive rate reference threshold of the CD45RO +, the first reference probability of the corresponding 5-year survival rate is 81%, and when the cell positive rate is less than the cell positive rate reference threshold of the CD45RO +, the second reference probability of the corresponding 5-year survival rate is 51%; when the cell positive rate of the CD8+ CD45RO + is greater than or equal to the cell positive rate reference threshold of the CD8+ CD45RO +, the first reference probability of the corresponding 5-year survival rate is 85%, and when the cell positive rate is less than the cell positive rate reference threshold of the CD8+ CD45RO +, the second reference probability of the corresponding 5-year survival rate is 68%; when the cell positive rate of the CD3+ PD1+ is greater than or equal to the cell positive rate threshold value of the CD3+ PD1+, the first reference probability of the corresponding 5-year survival rate is 100%, and when the cell positive rate of the CD3+ PD1+ is less than the cell positive rate threshold value, the second reference probability of the corresponding 5-year survival rate is 68%; when the cell positive rate of the CD8+ PD1+ is greater than or equal to the cell positive rate reference threshold of the CD8+ PD1+, the first reference probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate of the CD8+ PD1+ is less than the cell positive rate reference threshold, the second reference probability of the corresponding 5-year survival rate is 69%; when the cell positive rate of the CD4+ is greater than or equal to the cell positive rate reference threshold of the CD4+, the first reference probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate is less than the cell positive rate reference threshold of the CD4+, the second reference probability of the corresponding 5-year survival rate is 66%; when the cell positivity of the CD4+ FOXP3+ is greater than or equal to the cell positivity reference threshold of the CD4+ FOXP3+, the first reference probability of the corresponding 5-year survival rate is 83%, and when the cell positivity is less than the cell positivity reference threshold of the CD4+ FOXP3+, the second reference probability of the corresponding 5-year survival rate is 72%; when the cell positive rate of the CD68+ is greater than or equal to the cell positive rate reference threshold of the CD68+, the first reference probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate is less than the cell positive rate reference threshold of the CD68+, the second reference probability of the corresponding 5-year survival rate is 67%; when the cell positive rate of CD68+ CD 163-is greater than or equal to the cell positive rate reference threshold of CD68+ CD163-, the first reference probability of the corresponding 5-year survival rate is 93%, and when the cell positive rate of CD68+ CD 163-is less than the cell positive rate reference threshold, the second reference probability of the corresponding 5-year survival rate is 66%; when the cell positive rate of CD68+ CD163+ is greater than or equal to the cell positive rate reference threshold of CD68+ CD163+, the first reference probability of the corresponding 5-year survival rate is 81%, and when the cell positive rate is less than the cell positive rate reference threshold of CD68+ CD163+, the second reference probability of the corresponding 5-year survival rate is 66%; when the cell positivity of PDL + is greater than or equal to the cell positivity reference threshold of PDL +, the first reference probability of the corresponding 5-year survival rate is 72%, and when the cell positivity of PDL + is less than the cell positivity reference threshold of PDL +, the second reference probability of the corresponding 5-year survival rate is 81%; when the cell positive rate of CD57+ is equal to or higher than the cell positive rate reference threshold of CD57+, the first reference probability of the corresponding 5-year survival rate is 100%, and when the cell positive rate is lower than the cell positive rate reference threshold of CD57+, the second reference probability of the corresponding 5-year survival rate is 70%.
Further, the apparatus further comprises: the acquisition module is used for acquiring the POLE gene mutation detection result of the sample to be detected and the result indexes of the multiple immunohistochemical detection, wherein the result indexes comprise the following indexes: (a) cellular positivity rate of CD45RO + and at least one of the following molecular markers in intraepithelial, upon mutation of the ble gene: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, PD-L1+, and CD4 +; (b) intraepithelial, CD45RO + and the cellular positivity of at least one of the following molecular markers: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, CD8+ PD1+, CD4+, CD4+ FOXP3+, CD68+, CD68+ CD163+, CD68+ CD163-, PDL1+, and CD57 +; and the comparison module is used for comparing the result index with the first reference index and/or the second reference index.
Further, the device further comprises an output module for outputting the result index as a third reference index when the result index is consistent with the first reference index and/or the second reference index, or outputting the result index as a fourth reference index.
By applying the technical scheme of the invention, the invention discovers that the POLE gene mutation is obviously related to the high expression of the partial molecular markers, and determines the relation between the POLE gene mutation and the colorectal cancer tumor microenvironment, so that the advantages of high detection sensitivity and good specificity in the aspect of detecting the tumor microenvironment of a pancreatic patient and the simultaneous marking of a plurality of molecular markers by comprehensively utilizing a multiple immunohistochemical method are facilitated, and whether a detected object has a high-probability 5-year survival rate or not is relatively accurately predicted.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows the cell-positive rate of each molecular marker in the case where the POLE gene is a wild type and a mutant type; and
FIGS. 2A and 2B show the multiplex immunohistochemistry results for panel1 and panel2 in example 1 of the present application.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
Treg: regulatory T cells, Regulatory cells, Tregs for short. Are a subset of T cells that control autoimmune reactivity in vivo. Can be divided into naturally occurring regulatory T cells (n T-regs) and induced adaptive regulatory T cells (i T-regs). Regulatory T cells are associated with a variety of autoimmune diseases such as rheumatoid arthritis, autoimmune thyroiditis, autoimmune liver disease, and various kidney diseases.
MDSC: myeloid-derived suppressor cells, a heterogeneous population of cells derived from bone marrow, are precursors of dendritic cells, macrophages and granulocytes, and have the ability to significantly suppress immune cell responses.
As mentioned in the background art, there is no scheme for effectively detecting tumor immune microenvironment of colorectal cancer by adopting multiple immunohistochemical methods in the prior art, and to improve the current situation, the inventors have studied and screened the existing immune cells and immune checkpoint molecules of colorectal cancer, and found that the following 11 detection molecules can effectively identify colorectal cancer. Specific immune cells and immune checkpoint molecules include: CD3(T cell marker), CD8(CTL cell marker), CD45RO (memory T cell marker), CD4(T cell marker, e.g. Th, Treg, etc.), CD68 (macrophage marker), CD163(M2 type macrophage marker), FoxP3 (mainly Treg cell marker), CD57 (natural killer cell marker), PDL1 (immune checkpoint molecule), PD1 (immune checkpoint molecule) total 10 detection molecules.
Further, the inventor optimally combines the labeling effects of the 10 detection molecules on the colorectal cancer, and finds that the effect is better when the panel (combination) formed by combining the detection molecules in the following way is subjected to multiple immunohistochemical detection.
Preferred panel combinations are as follows:
Panel 1：CD3、CD8、CD45RO、PD-1、PD-L1
Panel 2：CD4、FOXP3、CD68、CD163、PD-L1、CD57。
by performing multiple immunohistochemical analysis detection on the point gene mutation and the combined molecular markers of 120 CRC samples in the II stage, the inventors found that: the POLE gene mutation is related to a colorectal cancer immune microenvironment, and the POLE gene mutation is remarkably related to high expression of intraepithelial CD3+, CD8+, CD45RO +, CD8+ CD45RO +, CD3+ PD1+, CD4+ and PD-L1+ in colorectal cancer tissues. Thus, using the above markers, in combination with the mutation in the pool gene, can collectively direct the patient to receive therapeutic benefit of immunotherapy (see in particular figure 1 and table 1, the horizontal axis in FIG. 1 represents the subpopulations of immune cells in the epithelium, and the vertical axis represents the percentage of cells, for a total of 120 CRC patients, wherein, the POLE mutant is 20 cases, the wild type is 100 cases, M1macrophage represents: CD68+ CD 163-macrophages; m2 macrophage indicates macrophages of CD68+ CD163+, NK cells indicate CD7+ cells, PDL + cells indicate the expression of PDL + cells in all the cells examined, and PDL + macrophages refer to PDL + expression in macrophages; table 1 shows that in the case of the poll wild type and mutant, the same words or terms are used herein to denote the same meaning.
Further, the inventors found that, from 120 cases of CRC samples in stage II, intraepithelial CD3+, CD8+, CD45RO +, CD8+ CD45RO +, CD3+ PD1+, CD4+, CD68+, CD68+ CD163-, CD57+ were highly expressed, and the 5-year survival rate of patients was high (see table 2 and table 3 in particular).
Therefore, the multiple immunohistochemistry simultaneous labeling of the multiple immune cell markers, the tumor cell markers and the immune checkpoint molecules is very beneficial for predicting whether the application of the immune checkpoint inhibitor medicine to the colorectal cancer patients is effective or not.
On the basis of the above research results, the applicant proposed the technical solution of the present application. In an exemplary embodiment of the present application, a colorectal cancer tumor microenvironment detection reagent is provided, which comprises a poll gene mutation detection reagent and an antibody of at least one molecular marker in at least one combination of the following:
combination 1: CD3, CD8, CD45RO, PD-1 and PD-L1;
and (3) combination 2: CD4, FOXP3, CD68, CD163, PD-L1 and CD 57.
The application finds that the POLE gene mutation is obviously related to the high expression of the partial molecular markers, and the relationship between the POLE gene mutation and the colorectal cancer tumor microenvironment is determined, so that the multiple immunohistochemical methods are conveniently and comprehensively utilized to simultaneously mark a plurality of molecular markers, the detection sensitivity is high in the aspect of detecting the tumor microenvironment of a pancreatic patient, the advantage of good specificity is realized, and the 5-year survival rate of a detected object with higher probability is relatively accurately predicted.
When detecting, the appropriate molecular markers can be selected according to actual needs for combined use. Preferably, the combination is performed on the principle of differentiating between different cell subsets and corresponding to relevant immune checkpoints. In the present application, the inventors found that the mutation of the pool gene is significantly related to the high expression of CD8+ T cells, CD45RO + T memory cells and CD8+ CD45RP + double positive cells in colorectal cancer, and therefore, in a preferred embodiment of the present application, the detection reagent comprises at least an antibody against CD45RO, more preferably an antibody against CD45RO and an antibody against CD8, and further preferably comprises at least antibodies against the following molecular markers: CD3, CD8, CD45RO, PD1, PD-L1 and CD 4.
When the antibody of the molecular marker provided by the application is used for detecting the colorectal cancer, the antibody is usually used as a primary antibody for identifying the molecular marker, and a secondary antibody with a detectable label is usually used for specifically identifying the secondary antibody for labeling and detecting, so that the purpose of detecting the molecular marker is achieved. Thus, in a preferred embodiment, the antibody to the molecular marker is labeled as a primary antibody and the detection reagent further comprises a secondary antibody, preferably selected from horseradish peroxidase, PV-6001, PV-6002 or PV-8000.
The detection reagent may contain a luminescent reagent used in combination with the secondary antibody, depending on the type of the secondary antibody used. In a preferred embodiment, the detection reagent further comprises a luminescent reagent, preferably, the luminescent reagent is selected from the group consisting of tyramine salts with fluorescent groups, 520-FITC, 540-AF517, 570-Cy3, 620-Cy3.5, 650-Cy5, and 690-Cy5.
In a second exemplary embodiment of the present application, a colorectal cancer tumor microenvironment detection kit is provided, and the kit comprises any one of the detection reagents. The kit adopting the detection reagent has the advantages of high sensitivity and good specificity for detecting the colorectal cancer, and can display the tumor microenvironment condition of the colorectal cancer on the whole.
The POLE gene and the relation between the POLE gene and the tumor immunity related molecular marker can be used for preparing any related reagent or product capable of detecting the colorectal cancer tumor microenvironment. In a third exemplary embodiment of the present application, there is provided a use of the above-mentioned poll gene mutation detection reagent and the antibody against at least one molecular marker in any one of the above-mentioned combinations in the preparation of a kit for detecting a microenvironment associated with colorectal cancer tumor.
In a preferred embodiment, the antibody to the molecular marker comprises an antibody to CD45 RO; preferably an antibody comprising CD8 and an antibody comprising CD45 RO; more preferably an antibody comprising at least the following molecular markers: CD3, CD8, CD45RO, PD1, PD-L1 and CD 4.
In a preferred embodiment, the above application comprises: detecting whether the POLE gene is mutated or not by using a POLE gene mutation detection reagent; and detecting the molecular marker by adopting a multiple immunohistochemistry method, preferably detecting the cell positive rate of CD45RO +, more preferably detecting the cell positive rate of CD8+ CD45RO +; further preferably, the cell positive rate of the following molecular markers is detected: CD3+, CD8+, CD45RO +, CD8+ CD45RO +, CD3+ PD1+, PD-L1+, and CD4 +.
In a more preferred embodiment, in the case where the able gene is the mutant type, it is detected whether or not the cell positive rate of the molecular marker is equal to or greater than the value of the cell positive rate shown in table 1.
Table 1:
| POLE genotype | Wild type | Mutant forms |
| Percentage of positive CD8+ cells/% | 0.33 | 2.78 |
| CD45RO + cell positive rate/%) | 1.33 | 4.61 |
| Percentage of positive CD8+ CD45RO + cells/% | 0.03 | 0.30 |
| Percentage of positive CD3+ cells/% | 1.08 | 5.83 |
| Percentage of positive CD3+ PD1+ cells/% | 0.11 | 0.75 |
| Percentage of positive CD4+ cells/% | 1.10 | 6.42 |
| Percentage of PD-L1 cell positive rate% | 1.12 | 2.24 |
Mutation of the POLE gene and loss-of-function mutation of POLE are related to polyposis and colorectal cancer. In colon, rectal and endometrial cancers, several hot spot mutations in the pool exonuclease domain were identified to affect their proofreading ability and result in ultra-high mutation rates. According to the multicolor IHC technology, expression of a marker related to a colorectal cancer immune microenvironment is detected, and the POLE gene mutation is found to be related to the colorectal cancer immune microenvironment, and is obviously related to high expression of CD3+, CD8+, CD45RO +, CD8+ CD45RO +, CD3+ PD1+, CD4+ and PD-L1+ in colorectal cancer. The subjects detect the above markers, and combine with the mutation of the POLE gene to instruct the patients to receive the curative effect of immunotherapy (see figure 1)
The method for detecting colorectal cancer by using the above molecular markers is not particularly limited, and any method capable of detecting colorectal cancer by using the molecular markers can be applied to the preparation process of the kit. In a preferred embodiment, the application includes detection by an immunohistochemical method, and in this case, other reagents related to the immunohistochemical detection method, such as antibodies against the molecular markers, secondary antibodies against the antibodies, luminescent reagents, and the like, can also be included in the kit.
In a preferred embodiment, the immunohistochemistry is multiplex immunohistochemistry, and the colorectal cancer sample is detected by a multiplex immunohistochemistry mode through the combination of the molecular markers, so that the advantages of multiplex immunohistochemistry detection can be fully exerted, and the advantages of high sensitivity and high specificity of the molecular markers can be fully utilized, and the detection result of the colorectal cancer is more accurate.
In a preferred embodiment, the above applications include statistical cellular positivity rates of intraepithelial CD45RO + and at least one of the following molecular markers: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, CD8+ PD1+, CD4+, CD4+ FOXP3+, CD68+, CD68+ CD163+, CD68+ CD163-, PDL1+, and CD57 +; preferably, whether the cell positive rate of each molecular marker is greater than or equal to the corresponding threshold value is counted, if so, the 5-year productivity of the detection sample is defined as a first probability, otherwise, the 5-year productivity of the detection sample is defined as a second probability, and the first probability is greater than the second probability.
In a more preferred embodiment, the threshold value of the cell positivity of each of the molecular markers is shown in table 2 below, and a value equal to or greater than the corresponding threshold value indicates a high 5-year survival rate and is represented as a first probability, and a value smaller than the corresponding threshold value indicates a low 5-year survival rate and is represented as a second probability.
Table 2:
| item | Threshold value of cell Positive Rate (%) |
| CD3+ T cells | 1.313％ |
| CD8+ T cells | 0.876％ |
| CD45RO + cells | 0.360％ |
| CD8+ CD45RO + T cells | 0.017％ |
| CD3+ PD1+ T cells | 0.246％ |
| CD8+ PD1+ T cells | 0.019％ |
| CD4+ T cells | 1.775％ |
| CD4+ FOXP3+ cells | 0.262％ |
| CD68+ macrophages | 0.355％ |
| M1macrophage cell | 0.208％ |
| M2 macrophage cell | 0.005％ |
| PDL1+ macrophages | 0.003％ |
| NK cells | 0.490％ |
| PDL1+ cells | 0.021％ |
Dividing the cases into two groups of high 5-year survival rate and low 5-year survival rate according to whether the cell positive rate of each molecular marker is higher than the threshold value of the cell positive rate of each molecular marker, and counting the probability of the 5-year survival rate of the two groups of cases under the condition of the positive rate of each molecular marker. In a preferred embodiment, the 5-year survival probabilities for the two groups of cases at each molecular index are shown in Table 3.
Table 3:
it should be noted that the specific values of the first probability and the second probability of 5-year survival rate may have a slight difference according to the number of cases to be studied, but fall into two different categories.
In the above preferred embodiments, depending on the specific molecular marker to be detected, if the positive rate of the cells is higher than the corresponding threshold, the survival time of the test subject is predicted to be longer, and the prognosis effect is better. Specifically, for different CRC samples, the more the number of indexes is according to the number of indexes detected by actual samples, the more accurate the comprehensive evaluation of the survival time and the prognosis effect is.
In a fourth exemplary embodiment of the present application, a colorectal cancer tumor microenvironment detection device is provided, which has a colorectal cancer detection molecule embedded therein, and the colorectal cancer detection molecule detects at least one molecular marker including a able gene and at least one of the following combinations: combination 1: CD3, CD8, CD45RO, PD-1 and PD-L1; and (3) combination 2: CD4, FOXP3, CD68, CD163, PD-L1 and CD 57.
The device provided by the application detects the tumor microenvironment of colorectal cancer by utilizing whether the POLE gene is mutated or not and any one or more molecular markers in the combination, particularly utilizes the correlation between PLEO gene mutation and high expression of CD3+, CD8+, CD45RO +, CD8+ CD45RO +, CD3+ PD1+, PD-L1+ and CD4+ cells, and comprehensively utilizes a multiple immunohistochemistry method to simultaneously mark the multiple immune cell markers, the tumor cell markers and the immune checkpoint molecules, so that the device has the advantages of high detection sensitivity and good specificity in the aspect of detecting the tumor microenvironment of pancreatic patients, and can relatively accurately predict the prognosis curative effect.
In a preferred embodiment, the device incorporates a first reference marker, and in the case of a mutation in the point gene, the first reference marker comprises the molecular marker CD45RO + and a reference cell positivity for at least one of the following molecular markers: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, PD-L1+, and CD4 +; preferably, the reference cell positive rate of CD45RO + is not less than 4.61%, the reference cell positive rate of CD8+ is not less than 2.78%, the reference cell positive rate of CD8+ CD45RO + is not less than 0.30%, the reference cell positive rate of CD3+ is not less than 5.83%, the reference cell positive rate of CD4+ is not less than 6.42%, the reference cell positive rate of CD3+ PD1+ is not less than 0.75%, and the reference cell positive rate of PD-L1+ is not less than 2.24%.
In a preferred embodiment, the device further comprises a second reference index, wherein the second reference index is any one or more of the following cell positive rate reference threshold values under endothelium: a cell-positivity reference threshold of CD3+, a cell-positivity reference threshold of CD8+, a cell-positivity reference threshold of CD45RO +, a cell-positivity reference threshold of CD8+ CD45RO +, a cell-positivity reference threshold of CD3+ PD1+, a cell-positivity reference threshold of CD8+ PD1+, a cell-positivity reference threshold of CD4+, a cell-positivity reference threshold of CD4+ FOXP3+, a cell-positivity reference threshold of CD68+, a cell-positivity reference threshold of CD68+ CD163-, a cell-positivity reference threshold of CD68+ CD163+, and a cell-positivity reference threshold of PDL1 +; preferably, the reference threshold value of the cell positivity of CD3+ is not less than 1.313%, the reference threshold value of the cell positivity of CD8+ is not less than 0.876%, the reference threshold value of the cell positivity of CD45RO + is not less than 0.360%, the reference threshold value of the cell positivity of CD8+ CD45RO + is not less than 0.017%, the reference threshold value of the cell positivity of CD3+ PD1+ is not less than 0.246%, the reference threshold value of the cell positivity of CD8+ PD1+ is not less than 0.019%, the reference threshold value of the cell positivity of CD4+ is not less than 1.775%, the reference threshold value of the cell positivity of CD4+ FOXP3+ is not less than 0.262%, the reference threshold value of the cell positivity of CD68+ is not less than 0.355%, the reference threshold value of the cell positivity of CD68+ CD 163-is not less than 0.208%, the reference threshold value of the cell positivity of CD68+ CD163+ is not less than 0.005%, and the.
In a preferred embodiment, the apparatus further comprises a third reference index and a fourth reference index, wherein the third reference index is a first reference probability of 5-year survival rate corresponding to a case where the cell positivity of any one or more of the following molecular markers is equal to or higher than a cell positivity reference threshold of the corresponding molecular marker, and the fourth reference index is a second reference probability of 5-year survival rate corresponding to a case where the cell positivity of any one or more of the following molecular markers is equal to or higher than a cell positivity reference threshold of the corresponding molecular marker:
when the cell positive rate of the CD3+ is greater than or equal to the cell positive rate reference threshold of the CD3+, the first reference probability of the corresponding 5-year survival rate is 89%, and when the cell positive rate is less than the cell positive rate reference threshold of the CD3+, the second reference probability of the corresponding 5-year survival rate is 64%;
when the cell positive rate of the CD8+ is greater than or equal to the cell positive rate reference threshold of the CD8+, the first reference probability of the corresponding 5-year survival rate is 100%, and when the cell positive rate is less than the cell positive rate reference threshold of the CD8+, the second reference probability of the corresponding 5-year survival rate is 67%;
when the cell positive rate of the CD45RO + is greater than or equal to the cell positive rate reference threshold of the CD45RO +, the first reference probability of the corresponding 5-year survival rate is 81%, and when the cell positive rate is less than the cell positive rate reference threshold of the CD45RO +, the second reference probability of the corresponding 5-year survival rate is 51%;
when the cell positive rate of the CD8+ CD45RO + is greater than or equal to the cell positive rate reference threshold of the CD8+ CD45RO +, the first reference probability of the corresponding 5-year survival rate is 85%, and when the cell positive rate is less than the cell positive rate reference threshold of the CD8+ CD45RO +, the second reference probability of the corresponding 5-year survival rate is 68%;
when the cell positive rate of the CD3+ PD1+ is greater than or equal to the cell positive rate threshold value of the CD3+ PD1+, the first reference probability of the corresponding 5-year survival rate is 100%, and when the cell positive rate of the CD3+ PD1+ is less than the cell positive rate threshold value, the second reference probability of the corresponding 5-year survival rate is 68%;
when the cell positive rate of the CD8+ PD1+ is greater than or equal to the cell positive rate reference threshold of the CD8+ PD1+, the first reference probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate of the CD8+ PD1+ is less than the cell positive rate reference threshold, the second reference probability of the corresponding 5-year survival rate is 69%;
when the cell positive rate of the CD4+ is greater than or equal to the cell positive rate reference threshold of the CD4+, the first reference probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate is less than the cell positive rate reference threshold of the CD4+, the second reference probability of the corresponding 5-year survival rate is 66%;
when the cell positivity of the CD4+ FOXP3+ is greater than or equal to the cell positivity reference threshold of the CD4+ FOXP3+, the first reference probability of the corresponding 5-year survival rate is 83%, and when the cell positivity is less than the cell positivity reference threshold of the CD4+ FOXP3+, the second reference probability of the corresponding 5-year survival rate is 72%;
when the cell positive rate of the CD68+ is greater than or equal to the cell positive rate reference threshold of the CD68+, the first reference probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate is less than the cell positive rate reference threshold of the CD68+, the second reference probability of the corresponding 5-year survival rate is 67%;
when the cell positive rate of CD68+ CD 163-is greater than or equal to the cell positive rate reference threshold of CD68+ CD163-, the first reference probability of the corresponding 5-year survival rate is 93%, and when the cell positive rate of CD68+ CD 163-is less than the cell positive rate reference threshold, the second reference probability of the corresponding 5-year survival rate is 66%;
when the cell positive rate of CD68+ CD163+ is greater than or equal to the cell positive rate reference threshold of CD68+ CD163+, the first reference probability of the corresponding 5-year survival rate is 81%, and when the cell positive rate is less than the cell positive rate reference threshold of CD68+ CD163+, the second reference probability of the corresponding 5-year survival rate is 66%;
when the cell positivity of PDL + is greater than or equal to the cell positivity reference threshold of PDL +, the first reference probability of the corresponding 5-year survival rate is 72%, and when the cell positivity of PDL + is less than the cell positivity reference threshold of PDL +, the second reference probability of the corresponding 5-year survival rate is 81%;
when the cell positive rate of CD57+ is equal to or higher than the cell positive rate reference threshold of CD57+, the first reference probability of the corresponding 5-year survival rate is 100%, and when the cell positive rate is lower than the cell positive rate reference threshold of CD57+, the second reference probability of the corresponding 5-year survival rate is 70%.
In the above preferred embodiments, the reference indexes of various biomarkers or their combinations are built in the detection device, which facilitates comparison and judgment of the corresponding indexes of the sample to be tested, thereby relatively accurately predicting the survival time and prognosis of the sample to be tested.
In a preferred embodiment, the above apparatus further comprises: the acquisition module is used for acquiring a POLE gene mutation detection result of a sample to be detected and result indexes of multiple immunohistochemical detections, wherein the result indexes comprise cell positive rates of (a) intraepithelial CD45RO + and at least one of the following molecular markers when the POLE gene is mutated: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, PD-L1+, and CD4 +; (b) intraepithelial, CD45RO + and the cellular positivity of at least one of the following molecular markers: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, CD8+ PD1+, CD4+, CD4+ FOXP3+, CD68+, CD68+ CD163+, CD68+ CD163-, PDL1+, and CD57 +; and the comparison module is used for comparing the result index with the first reference index and/or the second reference index.
In the above preferred embodiment, the device can obtain the poll gene mutation detection result and the related result index of the sample to be detected by simultaneously including the obtaining module and the comparing module, and compare the poll gene mutation detection result and the related result index with the first reference index and/or the second reference index by the comparing module, so that the comparison result can be directly displayed, and a user of the detection device can conveniently and directly obtain the comparison result.
In a preferred embodiment, the apparatus further comprises an output module for outputting the result indicator as a third reference indicator if the result indicator matches the first reference indicator and/or the second reference indicator, and otherwise as a fourth reference indicator.
The preferred embodiment further classifies the comparison results, and according to the difference of the comparison results, the samples to be tested are divided into two categories with different overall survival rates and/or disease-free survival rates. The specific values of the above indexes may have slight differences according to the number of the studied cases, but all belong to two different overall survival rates and disease-free survival rate ranges.
In the above preferred embodiments, which may be divided into two groups, one group is that the probability of predicting the 5-year survival rate of the subject is higher and the prognosis may be better when the positive rate of the cells is higher than the corresponding threshold. While the other group has a relatively low probability of 5-year survival and a relatively poor prognosis for its corresponding index below the corresponding threshold. Moreover, according to the number of indexes detected by the actual sample, the more the number of indexes is, the more accurate the comprehensive evaluation of the survival period and the prognosis effect is.
It should be noted that, among the molecular markers mentioned in the present application, the aforementioned molecular markers not specifically mentioned in role also have corresponding roles or potential roles in the tumor microenvironment detection of colorectal cancer, and are not described in detail herein.
The advantageous effects of the present application will be further described with reference to specific examples.
Example 1
Four patients in stage II were selected: 2 cases of rectal adenocarcinoma; colon mucinous adenocarcinoma 1 case; colon adenocarcinoma with mucinous adenocarcinoma 1 case, and specific information is shown in table 4.
Table 4:
| numbering | Tumor site | Clinical staging | T stage | Follow-up time (year) | Whether or not to die |
| 1 | Rectum and rectum | II | 4 | 6.7 | Whether or not |
| 2 | High position rectum | II | 3 | 4.0 | Whether or not |
| 3 | Sigmoid colon | II | 4 | 1.7 | Whether or not |
| 4 | Ascending colon | II | 4 | 1.2 | Is that |
The paraffin sections of the tumor tissues of the above 4 cases were subjected to multiplex immunohistochemical analysis for detection of immune cells and immune checkpoint molecules. 2 sections per patient, 2 stained panels in total, and 10 molecules labeled. The order of staining for each of the 10 stained molecules and 2 panels was: pane1, CD45RO, PD-L1, CD8, CD3, PD 1; panel 2: FoxP3, CD163, CD68, CD4, PDL1, CD 57.
Multiplex immunohistochemical analysis of immune cell and immune checkpoint molecular detection was performed as follows:
1. placing the paraffin section in a constant temperature box at 60 ℃ for baking for 120 minutes;
2. dewaxing and hydrating: xylene (10min) → absolute ethanol (5min × 2 times) → 95% ethanol (5min × 2) → 90% (5min) → 85% ethanol (5min) → 80% ethanol (5min) → 75% ethanol (5 min);
3. washing with distilled water for 2 times for 5 min;
4. antigen retrieval: by OpalTMCarrying out microwave repair on the antigen repair liquid in the 7-color fluorescent staining reagent kit for 5min, 2min at high fire and 15min at medium and low fire;
5. naturally cooling at room temperature;
6. washing: washing with TBST buffer solution for 3 times (5 min/time);
7. and (3) sealing: sealing with sealing liquid at room temperature for 10 min;
8. primary antibody incubation: dropping primary antibody (100-300 ul of primary antibody working solution),
the staining sequence for Panel1 was: CD45RO, PD-L1, CD8, CD3, PD1, wherein CD8 is incubated at 4 ℃ overnight and the remainder at 37 ℃;
the staining sequence for Panel2 was: FoxP3, CD163, CD68, CD4, PDL1, CD57, wherein CD68 and CD57 are incubated at 4 ℃ overnight and the remainder are incubated at 37 ℃ for 1 Hr;
9. washing: washing with TBST buffer solution for 3 times (5 min/time);
10. and (3) secondary antibody incubation: dripping the secondary antibody in the kit, and incubating for 10min at 37 ℃; the information for use of each Panel secondary antibody is detailed in table 2.
11. Washing: washing with TBST buffer solution for 3 times (5 min/time);
12. fluorescence development: dripping opal fluorescent staining after TSA dilution, and keeping the temperature at 10 min;
13. washing: washing with TBST buffer solution for 3 times (5 min/time);
14. antibody staining in sequence: after the first antibody is dyed, repeating the steps 4) to 13) for each subsequent antibody, and sequentially marking all the antibodies; the antibody staining sequence is detailed in step 8;
15. microwave treatment: repeating the steps 4) to 6);
dyeing with DAPI for 5-10 min at room temperature;
17. washing: TBST washing for 3 times, 5 min/time;
18. sealing: sealing a quenching agent prevention piece;
19. continuous spectrum imaging, image processing and observation analysis.
Table 5: 6 Panel primary antibody, secondary antibody and fluorescent dye information
According to the above operation steps, multiple markers of immune cell markers and immune checkpoint molecules for 4 colorectal cancer tumor tissue sections are completed. Continuous spectrum acquisition was performed using the Vectra system from PerkinElmer, Inc., and image processing and observation analysis were performed. In the process of spectrum collection, the marking colors of different marker molecules in each panel are respectively shown in table 6, and different pseudo colors are selected for signal collection. The final test results are shown in fig. 2A and 2B.
TABLE 6 Panel selection for false color
| Marker panel1 | CD3 | CD8 | CD45RO | PD1 | PD-L1 | - |
| Colour(s) | Magenta color | Green colour | Red colour | Cyan color | Orange yellow | - |
| Marker panel2 | CD4 | CD68 | CD163 | FoxP3 | PDL1 | CD57 |
| Colour(s) | Orange yellow | Magenta color | Green colour | Cyan color | Yellow colour | - |
Statistical analysis of each marker molecule in the epithelium (CT region) was performed, and the statistical results of the percentage positivity of immune cell markers and immune checkpoint molecules for 4 patients are shown in tables 7 and 8 below:
TABLE 7 statistics of percent positive expression for each molecule in the epithelium
| Cell positive rate/% | CD3+ | CD8+ | CD45RO+ | PD1+ | PDL1+ | CD8+CD45RO+ | CD8+PD1+ |
| 1 | 0.018 | 0.009 | 0.173 | 0 | 0.027 | 0 | 0 |
| 2 | 0.595 | 0.066 | 0.578 | 0.041 | 0.017 | 0 | 0 |
| 3 | 0 | 0.055 | 0.11 | 0.206 | 0.096 | 0 | 0 |
| 4 | 0.139 | 0.064 | 0.663 | 0.395 | 5.761 | 0 | 0.011 |
Table 8:
example 2
DNA samples of 120 CRC cases in the II stage are adopted to construct a whole genome sequencing library, and high-throughput sequencing is carried out to detect whether the POLE gene has mutation. The detection result is as follows: among 120 samples, 20 samples of the POLE gene mutant type always involved mutations at 20 SNP sites, and 100 samples of the POLE gene wild type.
The wild-type sample and the mutant sample of the POLE gene are divided into two groups, the expression of each molecular marker is detected by the same multiple immunohistochemical method as in example 1, and the expression of the cellular positivity of the immune microenvironment markers corresponding to the mutant and wild types of the POLE gene is detected. The results are shown in FIG. 1, and FIG. 1 shows the percentage of positive cells expressing different genotypes of POLE and each molecular marker or bimolecular marker.
Example 3
The cell positivity of each molecular marker in tumor intraepithelial (CT) was examined in 120 CRC stage II cases using the same multiplex immunohistochemical detection method as in example 1. The results of the measurements are shown in tables 2 and 3.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: when the detection reagent of the POLE gene, the molecular marker in any combination and the combination thereof provided by the application are used for detecting the colorectal cancer, the tumor microenvironment of a corresponding patient can be detected more accurately, so that the survival prognosis of a sample to be detected can be guided and predicted more effectively.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (15)
1. A colorectal cancer tumor microenvironment detection reagent, wherein the detection reagent comprises a POLE gene mutation detection reagent and an antibody of at least one molecular marker in at least one of the following combinations:
combination 1: CD3, CD8, CD45RO, PD-1 and PD-L1;
and (3) combination 2: CD4, FOXP3, CD68, CD163, PD-L1 and CD 57.
2. The detection reagent of claim 1, wherein the detection reagent comprises an antibody to at least a CD45RO molecular marker; preferably, the detection reagent comprises antibodies to at least two of the molecular markers CD8 and CD45 RO; more preferably, the detection reagent comprises antibodies to at least the following molecular markers: CD3, CD8, CD45RO, PD1, PD-L1 and CD 4.
3. The detection reagent according to claim 2, wherein the antibody of the molecular marker is a primary antibody, and the detection reagent further comprises a secondary antibody,
preferably, the secondary antibody is selected from horseradish peroxidase, PV-6001, PV-6002 or PV-8000;
preferably, the detection reagent further comprises a luminescent reagent, more preferably, the luminescent reagent is selected from tyramine salts with fluorescent groups, 520-FITC, 540-AF517, 570-Cy3, 620-Cy3.5, 650-Cy5, or 690-Cy5.5.
4. A colorectal cancer tumor microenvironment detection kit, wherein the kit comprises the colorectal cancer tumor microenvironment detection reagent of any one of claims 1 to 3.
Use of a POLE gene mutation detection reagent and an antibody to at least one molecular marker in at least one combination for the preparation of a kit related to the tumor microenvironment for the detection of colorectal cancer,
combination 1: CD3, CD8, CD45RO, PD-1 and PD-L1;
and (3) combination 2: CD4, FOXP3, CD68, CD163, PD-L1 and CD 57.
6. The use of claim 5, wherein the antibody to a molecular marker comprises an antibody to CD45 RO; preferably an antibody comprising CD8 and an antibody comprising CD45 RO; more preferably, the antibody comprises at least the following molecular markers: CD3, CD8, CD45RO, PD1, PD-L1 and CD 4.
7. The application according to claim 5 or 6, characterized in that it comprises:
detecting whether the POLE gene is mutated or not by using a POLE gene mutation detection reagent; and
the molecular markers are detected by a multiple immunohistochemical method, and preferably, the cell positive rate of at least two molecular markers is detected as follows: the cell positive rate of CD45RO +, the cell positive rate of CD8+, and the cell positive rate of CD8+ CD45RO +; more preferably, the cell positivity rate of CD45RO + and at least one of the following molecular markers is detected: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, PD-L1+, and CD4 +;
preferably, whether the cell positive rate of the CD45RO + is greater than or equal to 4.61%, whether the cell positive rate of the CD8+ is greater than or equal to 2.78%, whether the cell positive rate of the CD8+ CD45RO + is greater than or equal to 0.30%, whether the cell positive rate of the CD3+ is greater than or equal to 5.83%, whether the cell positive rate of the CD4+ is greater than or equal to 6.42%, whether the cell positive rate of the CD3+ PD1+ is greater than or equal to 0.75%, and whether the cell positive rate of the PD-L1+ is greater than or equal to 2.24% is detected in the case of the mutation of the POLE gene.
8. The use according to claim 5 or 6, characterized in that it comprises the statistical cellular positivity rate of intraepithelial CD45RO + and at least one of the following molecular markers: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, CD8+ PD1+, CD4+, CD4+ FOXP3+, CD68+, CD68+ CD163+, CD68+ CD163-, PDL1+, and CD57 +;
preferably, whether the cellular positive rate of each molecular marker is greater than or equal to a corresponding threshold value is counted, if so, the 5-year productivity of the detection sample is defined as a first probability, otherwise, the 5-year productivity of the detection sample is defined as a second probability, and the first probability is greater than the second probability;
more preferably, the threshold value of the cell positive rate of CD3+ is not less than 1.313%, the threshold value of the cell positive rate of CD8+ is not less than 0.876%, the threshold value of the cell positive rate of CD45RO + is not less than 0.360%, the threshold value of the cell positive rate of CD8+ CD45RO + is not less than 0.017%, the threshold value of the cell positive rate of CD3+ PD1+ is not less than 0.246%, the threshold value of the cell positive rate of CD8+ PD1+ is not less than 0.019%, the threshold value of the cell positive rate of CD4+ is not less than 1.775%, the threshold value of the cell positive rate of CD4+ FOXP3+ is not less than 0.262%, the threshold value of the cell positive rate of CD68+ is not less than 0.355%, the threshold value of the cell positive rate of CD68+ CD 163-is not less than 0.208%, the threshold value of the cell positive rate of CD68+ CD163+ is not less than 0.005%, and the threshold.
9. The use according to claim 8, further comprising counting said first probability of 5-year survival for each of said molecular markers being more than or equal to a respective threshold value of cellular positivity, or said second probability of 5-year survival for each of said molecular markers being less than a respective threshold value;
preferably, the first probability of 5-year survival is 89% when the cell positivity of CD3+ is greater than or equal to the corresponding threshold, and the second probability of 5-year survival is 64% when the cell positivity is less than the corresponding threshold;
said first probability of corresponding 5-year survival being 100% if said rate of cell positivity of CD8+ is greater than or equal to the respective threshold, and said second probability of corresponding 5-year survival being 67% if it is less than the respective threshold;
when the cell positive rate of the CD45RO + is greater than or equal to the corresponding threshold, the first probability of the corresponding 5-year survival rate is 81%, and when the cell positive rate is less than the corresponding threshold, the second probability of the corresponding 5-year survival rate is 51%;
when the cell positive rate of the CD8+ CD45RO + is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 85%, and when the cell positive rate of the CD8+ CD45RO + is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 68%;
said first probability of corresponding 5-year survival being 100% for a cell positivity of said CD3+ PD1+ greater than or equal to a respective threshold, and said second probability of corresponding 5-year survival being 68% for a cell positivity of said CD3+ PD1+ less than a respective threshold;
when the cell positive rate of the CD8+ PD1+ is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate of the CD8+ PD1+ is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 69%;
when the cell positive rate of the CD4+ is greater than or equal to the corresponding threshold, the first probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate is less than the corresponding threshold, the second probability of the corresponding 5-year survival rate is 66%;
when the cell positivity of the CD4+ FOXP3+ is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 83%, and when the cell positivity of the CD4+ FOXP3+ is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 72%;
when the cell positive rate of the CD68+ is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 67%;
said first probability of a corresponding 5-year survival rate is 93% when said cell positivity of CD68+ CD163 "is greater than or equal to a corresponding threshold value, and said second probability of a corresponding 5-year survival rate is 66% when said cell positivity of CD68+ CD 163" is less than a corresponding threshold value;
the first probability of a corresponding 5-year survival rate is 81% when the cell positive rate of CD68+ CD163+ is greater than or equal to the corresponding threshold value, and the second probability of a corresponding 5-year survival rate is 66% when the cell positive rate is less than the corresponding threshold value;
when the cell positive rate of the PDL + is greater than or equal to the corresponding threshold value, the first probability of the corresponding 5-year survival rate is 72%, and when the cell positive rate of the PDL + is less than the corresponding threshold value, the second probability of the corresponding 5-year survival rate is 81%;
said first probability of corresponding 5-year survival being 100% if said rate of cell positivity of CD57+ is greater than or equal to the respective threshold value, and said second probability of corresponding 5-year survival being 70% if it is less than the respective threshold value.
10. The colorectal cancer tumor microenvironment detection device is internally provided with colorectal cancer detection molecules, and the colorectal cancer detection molecules detect at least one molecular marker comprising a POLE gene and at least one of the following combinations:
combination 1: CD3, CD8, CD45RO, PD-1 and PD-L1;
and (3) combination 2: CD4, FOXP3, CD68, CD163, PD-L1 and CD 57.
11. The device of claim 10, wherein the device has a first reference indicator embedded therein,
in the case where the able gene is mutated, the first reference indicator comprises a reference cell positivity rate for the molecular marker CD45RO + and at least one of the following molecular markers: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, PD-L1+, and CD4 +;
preferably, the reference cell positive rate of the CD45RO + is greater than or equal to 4.61%, the reference cell positive rate of the CD8+ is greater than or equal to 2.78%, the reference cell positive rate of the CD8+ CD45RO + is greater than or equal to 0.30%, the reference cell positive rate of the CD3+ is greater than or equal to 5.83%, the reference cell positive rate of the CD4+ is greater than or equal to 6.42%, the reference cell positive rate of the CD3+ PD1+ is greater than or equal to 0.75%, and the reference cell positive rate of the PD-L1+ is greater than or equal to 2.24%.
12. The device of claim 11, further comprising a second reference indicator embedded therein,
the second reference index is any one or more of the following cell positive rate reference threshold values under endothelium: a cell-positivity reference threshold of CD3+, a cell-positivity reference threshold of CD8+, a cell-positivity reference threshold of CD45RO +, a cell-positivity reference threshold of CD8+ CD45RO +, a cell-positivity reference threshold of CD3+ PD1+, a cell-positivity reference threshold of CD8+ PD1+, a cell-positivity reference threshold of CD4+, a cell-positivity reference threshold of CD4+ FOXP3+, a cell-positivity reference threshold of CD68+, a cell-positivity reference threshold of CD68+ CD163-, a cell-positivity reference threshold of CD68+ CD163+, and a cell-positivity reference threshold of PDL1 +;
preferably, the reference threshold value of the cell positive rate of CD3+ is not less than 1.313%, the reference threshold value of the cell positive rate of CD8+ is not less than 0.876%, the reference threshold value of the cell positive rate of CD45RO + is not less than 0.360%, the reference threshold value of the cell positive rate of CD8+ CD45RO + is not less than 0.017%, the reference threshold value of the cell positive rate of CD3+ PD1+ is not less than 0.246%, the reference threshold value of the cell positive rate of CD8+ PD1+ is not less than 0.019%, the reference threshold value of the cell positive rate of CD4+ is not less than 1.775%, the reference threshold value of the cell positive rate of CD4+ FOXP3+ is not less than 0.262%, the reference threshold value of the cell positive rate of CD68+ is not less than 0.355%, the reference threshold value of the cell positive rate of CD68+ CD 163-is not less than 0.208%, the reference threshold value of the cell positive rate of CD68+ CD163+ is not less than 0.005%, and the reference threshold.
13. The device according to claim 12, further comprising a third reference index and a fourth reference index, wherein the third reference index is a first reference probability of 5-year survival rate corresponding to the case where the cell positivity of any one or more of the following molecular markers is greater than or equal to the cell positivity reference threshold of the corresponding molecular marker, and the fourth reference index is a second reference probability of 5-year survival rate corresponding to the case where the cell positivity of any one or more of the following molecular markers is greater than or equal to the cell positivity reference threshold of the corresponding molecular marker:
when the cell positive rate of the CD3+ is greater than or equal to the cell positive rate reference threshold of the CD3+, the first reference probability of the corresponding 5-year survival rate is 89%, and when the cell positive rate of the CD3+ is less than the cell positive rate reference threshold, the second reference probability of the corresponding 5-year survival rate is 64%;
when the cell positive rate of the CD8+ is greater than or equal to the cell positive rate reference threshold of the CD8+, the first reference probability of the corresponding 5-year survival rate is 100%, and when the cell positive rate of the CD8+ is less than the cell positive rate reference threshold, the second reference probability of the corresponding 5-year survival rate is 67%;
when the cell positive rate of the CD45RO + is greater than or equal to the cell positive rate reference threshold of the CD45RO +, the first reference probability of the corresponding 5-year survival rate is 81%, and when the cell positive rate of the CD45RO + is less than the cell positive rate reference threshold, the second reference probability of the corresponding 5-year survival rate is 51%;
when the cell positive rate of the CD8+ CD45RO + is greater than or equal to the cell positive rate reference threshold of the CD8+ CD45RO +, the first reference probability of the corresponding 5-year survival rate is 85%, and when the cell positive rate of the CD8+ CD45RO + is less than the cell positive rate reference threshold, the second reference probability of the corresponding 5-year survival rate is 68%;
when the cell positive rate of the CD3+ PD1+ is greater than or equal to the cell positive rate threshold of the CD3+ PD1+, the first reference probability of the corresponding 5-year survival rate is 100%, and when the cell positive rate of the CD3+ PD1+ is less than the cell positive rate threshold of the corresponding 5-year survival rate, the second reference probability of the corresponding 5-year survival rate is 68%;
when the cell positive rate of the CD8+ PD1+ is greater than or equal to the cell positive rate reference threshold of the CD8+ PD1+, the first reference probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate of the CD8+ PD1+ is less than the cell positive rate reference threshold, the second reference probability of the corresponding 5-year survival rate is 69%;
when the cell positive rate of the CD4+ is greater than or equal to the cell positive rate reference threshold of the CD4+, the first reference probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate of the CD4+ is less than the cell positive rate reference threshold, the second reference probability of the corresponding 5-year survival rate is 66%;
when the cell positivity of the CD4+ FOXP3+ is greater than or equal to the cell positivity reference threshold of the CD4+ FOXP3+, the first reference probability of the corresponding 5-year survival rate is 83%, and when the cell positivity reference threshold of the CD4+ FOXP3+ is less than, the second reference probability of the corresponding 5-year survival rate is 72%;
when the cell positive rate of the CD68+ is greater than or equal to the cell positive rate reference threshold of the CD68+, the first reference probability of the corresponding 5-year survival rate is 90%, and when the cell positive rate of the CD68+ is less than the cell positive rate reference threshold, the second reference probability of the corresponding 5-year survival rate is 67%;
the first reference probability of the corresponding 5-year survival rate is 93% when the cell positivity of the CD68+ CD 163-is greater than or equal to the cell positivity reference threshold of the CD68+ CD163-, and the second reference probability of the corresponding 5-year survival rate is 66% when the cell positivity of the CD68+ CD 163-is less than the cell positivity reference threshold;
when the cell positive rate of the CD68+ CD163+ is greater than or equal to the cell positive rate reference threshold of the CD68+ CD163+, the first reference probability of the corresponding 5-year survival rate is 81%, and when the cell positive rate of the CD68+ CD163+ is less than the cell positive rate reference threshold, the second reference probability of the corresponding 5-year survival rate is 66%;
when the cell positive rate of the PDL + is greater than or equal to the cell positive rate reference threshold value of the PDL +, the first reference probability of the corresponding 5-year survival rate is 72%, and when the cell positive rate of the PDL + is less than the cell positive rate reference threshold value of the PDL +, the second reference probability of the corresponding 5-year survival rate is 81%;
when the cell positive rate of the CD57+ is greater than or equal to the cell positive rate reference threshold of the CD57+, the first reference probability of the corresponding 5-year survival rate is 100%, and when the cell positive rate of the CD57+ is less than the cell positive rate reference threshold of the CD57+, the second reference probability of the corresponding 5-year survival rate is 70%.
14. The apparatus of claim 13, further comprising:
an obtaining module, configured to obtain a point gene mutation detection result of a sample to be detected and result indexes of multiple immunohistochemical detections, where the result indexes include, for example:
(a) cellular positivity rate of CD45RO + and at least one of the following molecular markers in intraepithelial, upon mutation of the ble gene: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, PD-L1+, and CD4 +;
(b) intraepithelial, CD45RO + and the cellular positivity of at least one of the following molecular markers: CD3+, CD8+, CD8+ CD45RO +, CD3+ PD1+, CD8+ PD1+, CD4+, CD4+ FOXP3+, CD68+, CD68+ CD163+, CD68+ CD163-, PDL1+, and CD57 +;
a comparison module to compare the result indicator with the first reference indicator and/or the second reference indicator.
15. The apparatus of claim 14, further comprising an output module configured to output the result indicator as the third reference indicator if the result indicator matches the first reference indicator and/or the second reference indicator, and to output the result indicator as the fourth reference indicator otherwise.
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| CN116086919A (en) * | 2023-02-20 | 2023-05-09 | 深圳裕策生物科技有限公司 | Staining method and kit for lung cancer and/or pancreatic cancer samples |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111778336A (en) * | 2020-07-23 | 2020-10-16 | 苏州班凯基因科技有限公司 | Gene marker combination for comprehensive quantitative evaluation of tumor microenvironment and application |
| CN111778336B (en) * | 2020-07-23 | 2021-02-26 | 苏州班凯基因科技有限公司 | Gene marker combination for comprehensive quantitative evaluation of tumor microenvironment and application |
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| CN113759132B (en) * | 2021-10-08 | 2022-06-03 | 首都医科大学附属北京妇产医院 | Models, products, and methods for predicting prognosis of endometrial cancer |
| CN116086919A (en) * | 2023-02-20 | 2023-05-09 | 深圳裕策生物科技有限公司 | Staining method and kit for lung cancer and/or pancreatic cancer samples |
| CN116086919B (en) * | 2023-02-20 | 2024-01-26 | 深圳裕策生物科技有限公司 | Staining method and kit for lung cancer and/or pancreatic cancer samples |
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