KIF11 and Ki67 immunohistochemical double staining kit and application thereof

Abstract

The invention relates to a KIF11 and Ki67 immunohistochemical double staining kit and application, and belongs to the technical field of biological medicine. The invention provides a KIF11 and Ki67 double-staining kit for tumor pathological typing and prognosis evaluation. The kit comprises a first monoclonal antibody of an anti-KIF11 protein, a second monoclonal antibody of an anti-Ki67 protein, and a third monoclonal antibody of an anti-Ki67 protein, a second monoclonal antibody of an anti-Ki67 protein; a first species-specific second antibody corresponding to the first monoclonal antibody, wherein the second antibody is coupled with a first enzyme; a second species-specific second antibody corresponding to the second monoclonal antibody, the second antibody being coupled with a second enzyme different from the first enzyme; a first chromogenic substrate corresponding to the first enzyme, wherein the first chromogenic substrate develops a color I; and the second chromogenic substrate corresponding to the second enzyme develops a second color which can be distinguished from the first color. According to the invention, an independent prognosis index is provided by quantifying the proportion of the special subgroup KIF11-High / Ki67-High cells, and the kit has an important prognosis value.

Description

Technical Field

[0001] This invention relates to a KIF11 and Ki67 immunohistochemical double staining kit and its application, belonging to the field of biomedical technology. Background Technology

[0002] Globally, the incidence and mortality rates of cancer are increasing year by year, seriously impacting human health. The high heterogeneity of tumors is a major reason for inconsistent treatment responses and vastly different prognoses. Therefore, developing diagnostic tools capable of accurately analyzing tumor heterogeneity and effectively stratifying risk is an urgent need in clinical practice.

[0003] The kinesin superfamily (KIF) is a family of molecular motors found in eukaryotes, playing crucial roles in a range of activities including cell transport, cell division, and microtubule cytoskeleton reorganization. This family comprises 14 subtypes and 45 kinesin genes. These molecular motors all share a characteristic motility domain composed of 350 amino acids. 【1】 Based on the location of their motor domains, they are further classified into N-kinesin (located at the amino terminus of the peptide chain, i.e., the N-terminus), M-kinesin (located at the amino terminus of the peptide chain, i.e., the M-terminus), and C-kinesin (located at the carboxyl terminus of the peptide chain, i.e., the C-terminus). N-kinesin is a motor protein that moves directionally towards the positive end of the microtubule, primarily responsible for intracellular transport, propelling microtubules to slide against each other, and regulating microtubule growth dynamics. M-kinesin does not move directionally but diffuses along the microtubule and can remove tubulin subunits from the microtubule tip, promoting microtubule depolymerization. Conversely, C-kinesin is a motor that moves towards the negative end of the microtubule; it can both cross-link microtubules and track and move towards the negative tip of the microtubule. Most of these are N-kinesin (kinesin 1-12), kinesin 13 is M-kinesin, and kinesin 14 is C-kinesin. 【2】Kinesin family member 11 (KIF11), also known as kinesin spindle protein (KSP) or kinesin-5 (Eg5), is a member of the kinesin family. This gene is located on human chromosome 10 at q23.33, contains 22 exons, and is 6216 kb in length. The protein encoded by this gene consists of 1057 amino acids and has a molecular weight of 119 kDa. In the cell, the mechanism of action of KIF11 protein is through the interaction of its stem domain, assembling into a bipolar homotetramer. This tetramer has two motility domains at each end, allowing it to simultaneously bind to two antiparallel microtubules and move along their positive ends, thereby pushing the two microtubules in opposite directions and promoting cell division. 【3-5】 Previous literature reports that KIF11 is closely related to the occurrence and development of various tumors, such as lung cancer, gastric cancer, pancreatic cancer, colorectal cancer, and ovarian cancer.

[0004] Ki67 is highly expressed in proliferating cells and is closely related to mitotic activity. It is currently used in clinical diagnosis to identify proliferating cell activity. This proliferation marker, initially discovered in the 1980s, is located at the q25 locus on chromosome 10 and contains 14 introns and 15 exons. 【6-7】 As is well known, Ki67 is closely related to the cell cycle. It is not expressed in the G0 phase, and its expression gradually increases in the G1, S, and G2 phases, but it cannot distinguish cells in the mitotic (M) phase. 【8】 KIF11 is a kinesiology protein that plays an irreplaceable role in the formation and maintenance of the mitotic spindle and is a key protein for the completion of the M phase of cell division. Therefore, KIF11 expression is a direct marker of mitotic activity in tumor cells.

[0005] Currently, KIF11 and Ki67 are typically detected by single staining on two separate tissue sections. This method cannot reveal the co-expression of both at the same cellular level, accurately identify the most aggressive cell subpopulations that are both in the proliferative cycle and highly mitotically active, or analyze the spatial distribution relationships of different subpopulations. Furthermore, two independent experiments increase sample consumption, reagent costs, and human error in interpretation. Therefore, there is an urgent need in the field for an immunohistochemical dual-staining kit that can simultaneously and accurately display KIF11 and Ki67 expression on a single tissue section to reveal more deeply the cell cycle heterogeneity of tumors and provide a more powerful tool for prognostic assessment and treatment decisions.

[0006] References

[0007] J Cell Biol.2004,167(1):19-22.doi:10.1083 / jcb.200408113【2】Hirokawa N,Noda Y,Tanaka Y,Niwa S.Kinesin superfamily motor proteins andintracellular transport.Nat Rev Mol Cell Biol.2009:10(10):682-696.doi:10.1038 / nrm2774

[0008] 【3】Rath O,Kozielski F.Kinesins and cancer.Nat Rev Cancer.2012:12(8):527-539.Published 2012Jul 24.doi:10.1038 / nrc3310

[0009] 【4】Bartoli KM,Jakovljevic J,Woolford JL Jr,Saunders WS.Functions of the kinesinmolecular motor Eg5 during polypeptide synthesis.Mol BiolCell.2011:22(18):3420-3430.doi:10.1091 / mbc.E11-03-0211

[0010] 【5】Liu M,Ran J,Zhou J.Non-canonical functions of the mitotic kinesinEg5.Thorac Cancer.2018:9(8):904-910.doi:10.1111 / 1759-7714.12792

[0011] 【6】Duchrow M, Schlüter C, Wohlenberg C, Flad HD, Gerdes J. Molecular characterization of the gene locus of the human cell proliferation-associated nuclear protein defined by monoclonal antibody Ki-67. Cell Prolif. 1996; 29(1):1-12.

[0012] 【7】Halm U,Tannapfel A,Breitung B,Breidert M,Wittekind CW, J. Apoptosis and cell proliferation in the metaplasia-dysplasia-carcinoma-sequence of Barrett's esophagus. Hepatogastroenterology. 2000; 47(34):962-966.

[0013] 【8】Gerlach C,Sakkab DY,Scholzen T,Dassler R,Alison MR,Gerdes J.Ki-67expression during rat liver regeneration after partialhepatectomy.Hepatology.1997;26(3):573-578.doi:10.1002 / hep.510260307 Summary of the Invention

[0014] One of the technical problems to be solved by this invention is how to prepare an immunohistochemical double staining kit that can simultaneously and accurately display the expression of KIF11 and Ki67 on the same slide and its application.

[0015] To address the aforementioned problems, the present invention provides a KIF11 and Ki67 double staining kit for tumor pathological classification and prognostic assessment, the kit comprising:

[0016] (1) The first monoclonal antibody against KIF11 protein;

[0017] (2) A second monoclonal antibody against Ki67 protein;

[0018] (3) A first genus-specific secondary antibody corresponding to the first monoclonal antibody, wherein the secondary antibody is conjugated with a first enzyme;

[0019] (4) A second genus-specific secondary antibody corresponding to the second monoclonal antibody, which is conjugated with a second enzyme different from the first enzyme;

[0020] (5) The first chromogenic substrate corresponding to the first enzyme develops color one;

[0021] (6) The second chromogenic substrate corresponding to the second enzyme develops a color that is distinguishable from color one.

[0022] Preferably, the first enzyme is horseradish peroxidase (HRP), the first chromogenic substrate is DAB / nickel salt enhancer, and the color is brownish-black; the second enzyme is alkaline phosphatase (AP), the second chromogenic substrate is Vector Green, FastGreen, or BCIP / NBT, and the color is green.

[0023] Preferably, the first enzyme is alkaline phosphatase (AP), the first chromogenic substrate is Vector Green, FastGreen, or BCIP / NBT, and the color is green; the second enzyme is horseradish peroxidase (HRP), the second chromogenic substrate is DAB / nickel salt enhancer, and the color is brownish-black.

[0024] Preferably, the kit further includes a buffer solution for antigen retrieval, blocking serum, washing solution, and sealing tablet.

[0025] This invention provides a method for double staining colorectal cancer tissue using the above-mentioned kit, comprising the following steps:

[0026] S1: Provide paraffin sections of tumor tissue and perform dewaxing and hydration;

[0027] S2: Perform antigen retrieval;

[0028] S3: Seal with sealing liquid;

[0029] S4: Simultaneously or sequentially add the first monoclonal antibody against KIF11 protein and the second monoclonal antibody against Ki67 protein, and incubate overnight at 4°C;

[0030] S5: After washing, add the corresponding first genus-specific secondary antibody and second genus-specific secondary antibody simultaneously or sequentially, and incubate at room temperature;

[0031] S6: After washing, add the first and second chromogenic substrates in sequence to carry out the color development reaction;

[0032] S7: Counterstain, dehydrate, clear and mount.

[0033] This invention provides the use of the above-described kit in the preparation of diagnostic products for tumor prognostic assessment.

[0034] Preferably, the tumor prognostic assessment includes the following steps:

[0035] S1: Use the above kit to stain tumor tissue samples;

[0036] S2: Observe and count at least 5 high-power fields of tumor cells under a microscope, and divide the cells into four subgroups based on the staining results: KIF11-High / Ki67-High, KIF11-High / Ki67-Low, KIF11-Low / Ki67-High, and KIF11-Low / Ki67-Low.

[0037] S3: Calculate the percentage of the four subgroups in the total tumor cells;

[0038] S4: When the percentage is higher than the preset threshold, the patient is determined to be a high-risk group with a poor prognosis;

[0039] The preset threshold value is the median value of the study cohort; the cutoff value for KIF11 / Ki67 double staining is 8%; patients are divided into a high-proportion group (≥8%) and a low-proportion group (<8%); the overall survival time and disease-free survival time of patients in the high-proportion group are significantly lower than those in the low-proportion group.

[0040] This invention provides the application of the above-described kit in the preparation of diagnostic products for differentiating cell cycle heterogeneity in colorectal cancer cells.

[0041] This invention provides the use of the above-described kit in the preparation of diagnostic products for prognostic assessment of colorectal cancer patients; the overall survival and disease-free survival of patients in the high proportion of the KIF11-High / Ki67-High group were significantly lower than those in the low proportion group.

[0042] This invention provides a detection system, including a data processing device and a substance for detecting the biomarker KIF11 / Ki67. The data processing device includes a data input module, a data recording module, a data comparison module, and a conclusion output module. The substance for detecting the biomarker includes the aforementioned reagent kit. The data input module is configured to input a biomarker detection image of a sample to be tested. The data recording module is configured to store the sample detection image and a judgment threshold. The data comparison module is configured to receive the biomarker detection image of the sample to be tested sent by the data input module, and retrieve the judgment threshold from the data recording module and compare it with the sample detection image. The conclusion output module is configured to receive the comparison result sent by the data comparison module, and judge the comparison result according to predetermined judgment conditions to assess the prognostic probability of the tested individual.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] This invention provides a simple, stable, and highly specific KIF11 and Ki67 immunohistochemical dual staining kit that can be used for precise pathological classification and prognostic assessment of tumors.

[0045] This invention enables precise subtyping: for the first time, tumor cells are divided into four functionally defined subgroups at the single-cell level, which intuitively reflects the cell cycle heterogeneity of tumors.

[0046] This invention has significant prognostic value: by quantifying the proportion of the special subset of KIF11-High / Ki67-High cells, it provides an independent prognostic indicator, overcoming the limitations of the traditional Ki67 index.

[0047] The kit of this invention has a standardized operating procedure, the results are easy to interpret (brown nucleus and green cytoplasm, brown nucleus / green cytoplasm, clear contrast), and good reproducibility, making it very suitable for widespread application in routine pathology departments.

[0048] This invention has broad application prospects: in principle, the kit can be applied to the research and diagnosis of any actively proliferating solid tumors (such as colorectal cancer, gastric cancer, breast cancer, lung cancer, etc.), providing a new perspective for the assessment of tumor biological behavior. Attached Figure Description

[0049] Figure 1 The images show representative results of KIF11-High / Ki67-High, KIF11-High / Ki67-Low, KIF11-Low / Ki67-High, and KIF11-Low / Ki67-Low double staining of colorectal cancer tissues in embodiments of the present invention.

[0050] Figure 2The proportions of different cell subpopulations with differential KIF11 / Ki67 expression in colorectal cancer tissue;

[0051] Figure 3 This is a Kaplan-Meier OS survival curve for patients in the KIF11-High / Ki67-High group in an embodiment of the present invention.

[0052] Figure 4 This is a Kaplan-Meier DFS survival curve for patients in the KIF11-High / Ki67-High group in an embodiment of the present invention. Detailed Implementation

[0053] To make the present invention more apparent and understandable, preferred embodiments are described in detail below:

[0054] Example 1

[0055] A KIF11 and Ki67 immunohistochemical double staining kit for tumor pathological classification and prognostic assessment was prepared. The kit includes:

[0056] (1) The first monoclonal antibody against KIF11 protein;

[0057] (2) A second monoclonal antibody against Ki67 protein;

[0058] (3) The first genus-specific secondary antibody corresponding to the first monoclonal antibody mentioned above, which is conjugated with the first enzyme;

[0059] (4) A second species-specific secondary antibody corresponding to the second monoclonal antibody mentioned above, which is conjugated with a second enzyme that is different from the first enzyme;

[0060] (5) The first chromogenic substrate corresponding to the first enzyme mentioned above;

[0061] (6) The second chromogenic substrate corresponding to the second enzyme mentioned above.

[0062] The first enzyme mentioned above is horseradish peroxidase (HRP), the first chromogenic substrate is DAB / nickel salt enhancer, and the color is brownish-black; the second enzyme mentioned above is alkaline phosphatase (AP), the second chromogenic substrate is Vector Green, Fast Green or BCIP / NBT, and the color is green.

[0063] Alternatively, the first enzyme mentioned above is alkaline phosphatase (AP), and the first chromogenic substrate is Vector Green, Fast Green, or BCIP / NBT, resulting in a green color; the second enzyme mentioned above is horseradish peroxidase (HRP), and the second chromogenic substrate is DAB / nickel salt enhancer, resulting in a brownish-black color.

[0064] The kit also includes buffer solution for antigen retrieval, blocking serum, washing solution, and sealing tablets.

[0065] Example 2

[0066] Double staining procedure and result interpretation of the reagent kit

[0067] 1. Materials: Paraffin-embedded tissue removed during surgery from colorectal cancer patients was prepared into 4μm thick sections.

[0068] 2. Staining: Using the kit of this invention, follow these steps:

[0069] a) Dewaxing and hydration.

[0070] b) Heat-induced antigen retrieval was performed using citrate buffer (pH 6.0).

[0071] c) 3% hydrogen peroxide blocks endogenous peroxidase, resulting in serum blockade.

[0072] d) Simultaneously add rabbit-derived anti-KIF11 monoclonal antibody and mouse-derived anti-Ki67 monoclonal antibody, and incubate overnight at 4°C.

[0073] e) After washing, add rabbit universal secondary antibody and incubate at room temperature for 30 minutes.

[0074] f) After washing, add DAB chromogenic substrate for Ki67 (brown) chromogenic development, and control the reaction time under a microscope.

[0075] After washing, Vector Green substrate was added for KIF11 (green) staining, and the reaction time was controlled under a microscope.

[0076] g) Hematoxylin counterstaining of the nucleus, dehydration, clearing, and mounting with neutral resin.

[0077] 3. Result Interpretation: For example... Figure 1 As shown, four cell subpopulations can be clearly distinguished under an optical microscope, with strong staining specificity and clear background. Tumor cells were observed and counted in at least five high-power fields under a microscope, and based on the staining results, the cells were divided into four subpopulations: KIF11-High / Ki67-High, KIF11-High / Ki67-Low, KIF11-Low / Ki67-High, and KIF11-Low / Ki67-Low.

[0078] like Figure 1The image shows representative results of KIF11-High / Ki67-High, KIF11-High / Ki67-Low, KIF11-Low / Ki67-High, and KIF11-Low / Ki67-Low double staining in colorectal cancer tissues. Labels 1 and 2 show cells with high KIF11-High / Ki67-High double expression (KIF11 is shown as green in the cytoplasm, and Ki67 as brown in the nucleus). Labels 3 and 4 show KIF11-High / Ki67-Low cells (more green, less brown). Labels 5 and 6 show KIF11-Low / Ki67-High cells (less green, more brown). Labels 7 and 8 show KIF11-Low / Ki67-Low cells (less green, less brown). The scale bars for HE and immunohistochemistry are 90 μm.

[0079] Example 3:

[0080] Application of reagent kits in prognostic assessment

[0081] 1. Cohort study: Tissue samples were collected from 266 colorectal cancer patients with complete 5-year follow-up data and double-stained using the kit of this invention.

[0082] 2. Quantitative Analysis:

[0083] Five high-power fields were counted for each sample, and KIF11 was calculated. + / Ki67 + Percentage of double-positive cells. Patients were divided into a high-proportion group (≥8%) and a low-proportion group (<8%), using the median (8%) as the cutoff value.

[0084] 3. Statistical analysis: Kaplan-Meier survival analysis showed (e.g.) Figure 3 , Figure 4 Patients in the high-proportion group had significantly lower overall survival and disease-free survival than those in the low-proportion group (Log-rank P < 0.05). Multivariate Cox regression analysis confirmed that the KIF11-High / Ki67-High group was an independent prognostic factor for colorectal cancer (OS, P = 0.012; DFS, P = 0.021). Using the pre-specified cutoff value as the median of the study cohort, the cutoff value for KIF11 monostaining in the KIF / Ki67 double staining group was 5%, the cutoff value for Ki67 monostaining was 28%, and the cutoff value for KIF11 / Ki67 double staining was 8%.

[0085] Univariate survival analysis showed that patients in the KIF11-High / Ki67-High group had shorter OS and DFS (P<0.05).

[0086] Table 1. Expression levels of KIF11 and Ki67 in 266 colorectal cancer cases.

[0087]

[0088] Table 2. Kaplan-Meier Survival Analysis of KIF11-Ki67 Dual Staining in Colorectal Cancer

[0089]

[0090] Table 3. Cox regression analysis of KIF11-Ki67 double staining and other clinicopathological parameters on colorectal cancer overall survival (OS).

[0091] Table 3.Cox regression analysis of KIF11-Ki67 and other clinicalpathological parameters for OS in Colorectal Cancer

[0092]

[0093] Table 4. Cox regression analysis of KIF11-Ki67 double staining and other clinicopathological parameters on disease-free survival (DFS) in colorectal cancer.

[0094] Table 4.Cox regression analysis of KIF11-Ki67 and other clinicalpathological parameters for DFS in Colorectal Cancer

[0095]

[0096] In summary, the KIF11 and Ki67 double staining kit provided by this invention can effectively identify cell subpopulations with high malignant potential in colorectal cancer, providing an important diagnostic tool for realizing personalized precision medicine.

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A KIF11 and Ki67 double staining kit for tumor pathological classification and prognostic assessment, characterized in that, The kit includes: (1) The first monoclonal antibody against KIF11 protein; (2) A second monoclonal antibody against Ki67 protein; (3) A first genus-specific secondary antibody corresponding to the first monoclonal antibody, which is conjugated with a first enzyme; (4) A second genus-specific secondary antibody corresponding to the second monoclonal antibody, which is conjugated with a second enzyme different from the first enzyme; (5) The first chromogenic substrate corresponding to the first enzyme develops color one; (6) The second chromogenic substrate corresponding to the second enzyme develops a color that is distinguishable from color one.

2. The KIF11 and Ki67 double staining kit for tumor pathological classification and prognostic assessment according to claim 1, characterized in that, The first enzyme is horseradish peroxidase (HRP), the first chromogenic substrate is DAB / nickel salt enhancer, and the color is brownish-black; the second enzyme is alkaline phosphatase (AP), the second chromogenic substrate is Vector Green, FastGreen, or BCIP / NBT, and the color is green.

3. The KIF11 and Ki67 double staining kit for tumor pathological classification and prognostic assessment according to claim 1, characterized in that, The first enzyme is alkaline phosphatase (AP), and the first chromogenic substrate is Vector Green, FastGreen, or BCIP / NBT, resulting in a green color. The second enzyme is horseradish peroxidase (HRP), and the second chromogenic substrate is DAB / nickel salt enhancer, resulting in a brownish-black color.

4. The KIF11 and Ki67 double staining kit for tumor pathological classification and prognostic assessment according to claim 1, characterized in that, The kit also includes a buffer solution for antigen retrieval, blocking serum, washing solution, and sealing tablets.

5. A method for double staining colorectal cancer tissue using a kit according to any one of claims 1-4, characterized in that, Includes the following steps: S1: Provide paraffin sections of tumor tissue and perform dewaxing and hydration; S2: Perform antigen retrieval; S3: Seal with sealing liquid; S4: Simultaneously or sequentially add the first monoclonal antibody against KIF11 protein and the second monoclonal antibody against Ki67 protein, and incubate overnight at 4°C; S5: After washing, add the corresponding first genus-specific secondary antibody and second genus-specific secondary antibody simultaneously or sequentially, and incubate at room temperature; S6: After washing, add the first and second chromogenic substrates in sequence to carry out the color development reaction; S7: Counterstain, dehydrate, clear and mount.

6. Use of the kit according to any one of claims 1 to 4 in the preparation of diagnostic articles for tumor prognostic assessment.

7. The use according to claim 6, characterized in that, The tumor prognostic assessment Includes the following steps: S1: Use the kit to stain tumor tissue samples; S2: Observe and count at least 5 high-power fields of tumor cells under a microscope, and divide the cells into four subgroups based on the staining results: KIF11-High / Ki67-High, KIF11-High / Ki67-Low, KIF11-Low / Ki67-High, and KIF11-Low / Ki67-Low. S3: Calculate the percentage of the four subgroups in the total tumor cells; S4: When the percentage is higher than the preset threshold, the patient is determined to be a high-risk group with a poor prognosis; the preset threshold is the median of the study cohort; the cutoff value of KIF11 / Ki67 double staining is 8%; the patients are divided into a high-proportion group (≥8%) and a low-proportion group (<8%); the overall survival time and disease-free survival time of the high-proportion group are significantly lower than those of the low-proportion group.

8. The use of the kit according to any one of claims 1 to 4 in the preparation of diagnostic products for differentiating cell cycle heterogeneity in colorectal cancer cells.

9. Use of the kit according to any one of claims 1 to 4 in the preparation of a diagnostic product for prognostic assessment of patients with colorectal cancer; characterized in that, Patients in the high proportion of the KIF11-High / Ki67-High group had significantly lower overall survival and disease-free survival than those in the low proportion group.

10. A detection system, characterized in that, The invention includes a data processing device and a substance for detecting the biomarker KIF11 / Ki67; the data processing device includes a data input module, a data recording module, a data comparison module, and a conclusion output module; the substance for detecting the biomarker includes the kit described in claims 1-4; the data input module is configured to input a biomarker detection image of a sample to be tested; the data recording module is configured to store the detection image of the sample to be tested and a judgment threshold; the data comparison module is configured to receive the biomarker detection image of the sample to be tested sent by the data input module, and retrieve the judgment threshold from the data recording module and compare it with the detection image of the sample to be tested; The conclusion output module is configured to receive the comparison result sent by the data comparison module, and to determine the comparison result according to predetermined judgment conditions to assess the prognosis of the tested person.

Images