Use of an agent that specifically binds to an oard1 protein in the manufacture of a product for the diagnosis and / or prognosis assessment of breast cancer

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a reagent specifically binding to OARD1 protein in preparation of a breast cancer diagnosis and / or prognosis evaluation product. The product detects the expression level of OARD1 protein in a breast tissue sample by the reagent specifically binding to OARD1 protein and carries out H-score scoring, takes a preset H-score score as a breast cancer positive diagnosis threshold, and judges whether the sample is breast cancer positive. Moreover, the product can distinguish high / low expression of OARD1 protein by a preset H-score score, constructs a prognosis prediction model in combination with TNM staging of a subject, and realizes evaluation of a breast cancer patient disease progression risk. The method is simple and easy to implement, the diagnosis process is safe and effective, is easy to be accepted by patients, diagnosis standards are unified, and is less affected by personal subjective factors, which shows that OARD1 protein has important significance for diagnosis or prognosis judgment of breast cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to the application of reagents that specifically bind to the OARD1 protein in the preparation of products for breast cancer diagnosis and / or prognostic assessment. Background Technology

[0002] Early diagnosis is crucial for improving breast cancer prognosis, but the current diagnostic system still faces many bottlenecks that hinder accurate early detection. Imaging examinations, as the mainstream screening method, each have significant limitations: breast ultrasound has a high rate of missed diagnoses for tiny tumors smaller than 0.1 cm in diameter and has difficulty identifying microcalcifications; some benign lesions and early-stage cancers share similar ultrasound characteristics, leading to misdiagnosis; mammography has a relatively higher rate of missed diagnoses in dense breasts (which are more common in Asian women) and carries radiation risks, making it unsuitable for routine screening in young women; while MRI has outstanding sensitivity, its false positive rate is also quite high, and the examination is costly and time-consuming, making it difficult to promote as a routine method.

[0003] Pathological biopsy, as the gold standard for diagnosis, also faces dual challenges in terms of technology and resources. Tumor heterogeneity may lead to the omission of key characteristic areas during sampling, and the concordance rate of diagnoses for the same case among different pathologists is lower than normal, with even lower concordance rates in cases of atypical hyperplasia. Manual diagnosis relies on physician experience and is prone to missing subtle lesions due to visual fatigue. The shortage of professional personnel in primary healthcare institutions leads to frequent diagnostic delays. Although new technologies such as liquid biopsy and ctDNA testing have shown potential, they have not yet achieved widespread clinical application due to issues such as extremely low levels of tumor markers, inconsistent testing standards, and high costs. The detection rate of stage I breast cancer is low, making it difficult to meet the needs of early diagnosis.

[0004] Furthermore, the difficulty some patients face in accessing medical care leads to late-stage diagnosis, hindering further improvements in breast cancer survival rates. Therefore, developing novel, highly sensitive, and specific tumor diagnostic and prognostic biomarkers to overcome existing technological limitations and build a precise, efficient, and accessible diagnostic system has become an urgent need in the field of breast cancer prevention and control. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides the application of reagents that specifically bind to the OARD1 protein in the preparation of products for breast cancer diagnosis and / or prognostic assessment. The method is simple and easy to perform, the diagnostic process is safe and effective, easily accepted by patients, and the diagnostic criteria are standardized, with minimal influence from individual subjective factors, demonstrating the significant role of the OARD1 protein in the diagnosis or prognosis of breast cancer.

[0006] To achieve the above objectives, the present invention provides, on the one hand, the application of a reagent that specifically binds to the OARD1 protein in the preparation of a breast cancer diagnostic product. The product uses the reagent that specifically binds to the OARD1 protein to detect the expression level of the OARD1 protein in breast tissue samples and to perform H-Score scoring (immunohistochemical scoring). A preset H-Score score is used as a positive diagnostic threshold for breast cancer to determine whether the sample is positive for breast cancer.

[0007] The second aspect of this invention provides the application of a reagent that specifically binds to the OARD1 protein in the preparation of a breast cancer prognostic assessment product. The product detects the expression level of the OARD1 protein in breast cancer tissue samples and performs H-Score scoring using a reagent that specifically binds to the OARD1 protein. The preset H-Score score distinguishes between high and low expression of the OARD1 protein. Combined with the subject's TNM stage, a prognostic prediction model is constructed to assess the risk of disease progression in breast cancer patients.

[0008] Furthermore, when the preset H-Score is 4.5, the diagnostic sensitivity is 93.3% and the specificity is 88.6%.

[0009] Furthermore, when H-Score > 4.5, the diagnostic accuracy is ≥ 90.48%.

[0010] Furthermore, when the preset H-Score is a median of 4, patients are grouped according to their H-Score: those above the median are in the high-expression group, and those below or equal to the median are in the low-expression group. In this case, the model's prediction formula is: Risk Score = 0.38 × Protein Expression Level Assignment + 0.62 × TNM Stage Assignment. When the risk score is ≥2.48, the 5-year survival rate of patients is ≤48.9%. Among them, the protein expression level assignment is: low expression = 0, high expression = 2; the TNM stage assignment is: stage I = 1, stage II = 2, stage III = 4, stage IV = 6.

[0011] Furthermore, the criteria for determining the H-Score are as follows:

[0012] The H-Score is calculated by combining the staining area score (percentage of positively stained area) and the staining intensity score in immunohistochemistry. The staining area score ranges from 0 to 4: 0 indicates no immunostaining, 1 indicates 1% to 14% positive, 2 indicates 15% to 49% positive, 3 indicates 50% to 74% positive, and 4 indicates 75% or more positive. The staining intensity score ranges from 0 to 4: 0 (no color), 1 (light yellow), 2 (light brown), 3 (brown), and 4 (dark brown). The H-Score for each tissue is calculated by adding the staining intensity score and the staining area score (therefore, the H-Score ranges from 0 to 8).

[0013] A third aspect of the present invention provides a kit for the diagnosis and / or prognostic assessment of breast cancer, comprising a reagent that specifically binds to the OARD1 protein; the reagent enables the detection of the OARD1 protein expression level in a sample by immunohistochemistry.

[0014] Furthermore, the kit also includes antigen retrieval solution, HRP-labeled secondary antibody, DAB chromogenic solution, positive control and negative control.

[0015] Furthermore, the conditions for the immunohistochemical staining include: primary antibody incubation temperature of 2℃~10℃ and incubation time of 1~16 hours.

[0016] Furthermore, the conditions for the immunohistochemical staining include: the secondary antibody incubation temperature is room temperature, and the incubation time is 10-60 minutes.

[0017] A fourth aspect of the present invention provides a breast cancer diagnosis and / or prognostic assessment system based on OARD1 protein expression levels, comprising:

[0018] The data acquisition module is used to acquire OARD1 protein-related data of subjects or to acquire OARD1 protein-related data and TNM staging information of patients; the OARD1 protein-related data is the H-Score score of OARD1 protein or OARD1 protein expression level data;

[0019] The analysis and processing module, which is communicatively connected to the data acquisition module, is used to perform at least one of the following operations:

[0020] (1) Diagnostic analysis: The H-Score of the OARD1 protein is compared with the positive diagnostic threshold for breast cancer described in this invention to generate a positive or negative diagnostic indicator for breast cancer;

[0021] (2) Prognostic analysis: Input the OARD1 protein expression level data and TNM staging information into the prognostic prediction model of the present invention to generate a prognostic indicator of the patient's 5-year survival risk;

[0022] The result output module is communicatively connected to the analysis and processing module and is used to output the diagnostic indication or prognosis indication.

[0023] Furthermore, the data acquisition module includes an immunohistochemical detection data interface for receiving the OARD1 protein H-Score obtained after immunohistochemical detection of breast tissue or breast cancer tissue samples.

[0024] Compared with the prior art, the present invention has at least the following beneficial effects:

[0025] (1) The present invention has discovered that OARD1 protein can be used for breast cancer diagnosis or prognostic assessment. According to the experiment of the present invention, the expression of OARD1 protein in breast cancer tissue is significantly higher than that in adjacent normal tissue, and it can be used as a biomarker for breast cancer diagnosis. Moreover, it shows that the expression of OARD1 protein is closely related to the TNM stage and survival of breast cancer patients.

[0026] (2) The experiment of this invention uses immunohistochemical staining to detect samples and confirms that OARD1 protein can be used as a molecular marker for the diagnosis of breast cancer. It is simple and easy to perform, the diagnostic process is safe and effective, it is easily accepted by patients, the diagnostic criteria are uniform, and it is less affected by personal subjective factors. This further shows that OARD1 protein is of great significance for the prognosis of breast cancer.

[0027] (3) In this invention, the OARD1 protein can be used as a biomarker for diagnosing breast cancer. When the immunohistochemical score H-Score of the OARD1 protein is 4.5, the sensitivity is 93.3% and the specificity is 88.6%. When performing individual testing, if the immunohistochemical score H-Score of the OARD1 protein is greater than 4.5, the patient is diagnosed with breast cancer with an accuracy of 90.48%. The OARD1 protein provided by this invention, as a diagnostic biomarker for breast cancer, improves sensitivity, specificity, and accuracy. Attached Figure Description

[0028] Figure 1 The results show the comparison of OARD1 expression in breast cancer and adjacent normal tissues, p < 0.001;

[0029] Figure 2 The correlation between OARD1 expression level and prognosis in breast cancer patients is shown in the figure, p=0.0002;

[0030] Figure 3 The results of the ROC analysis for predicting breast cancer incidence using OARD1 are shown. Detailed Implementation

[0031] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0032] According to one aspect of the present invention, a reagent that specifically binds to the OARD1 protein is used in the preparation of a breast cancer diagnostic product. The product detects the expression level of the OARD1 protein in a breast tissue sample and performs H-Score scoring using the reagent that specifically binds to the OARD1 protein. A preset H-Score is used as a breast cancer positive diagnostic threshold to determine whether the sample is positive for breast cancer.

[0033] In this invention, the OARD1 protein can be used as a biomarker for diagnosing breast cancer. When the immunohistochemical H-score of the OARD1 protein is 4.5, the sensitivity is 93.3% and the specificity is 88.6%. In individual testing, an OARD1 protein immunohistochemical H-score greater than 4.5 is considered indicative of breast cancer, with an accuracy of 90.48%. The OARD1 protein provided by this invention, as a diagnostic biomarker for breast cancer, improves sensitivity, specificity, and accuracy.

[0034] According to a second aspect of the present invention, a reagent that specifically binds to the OARD1 protein is used in the preparation of a breast cancer prognostic assessment product. The product detects the expression level of the OARD1 protein in breast cancer tissue samples and performs H-Score scoring using a reagent that specifically binds to the OARD1 protein. The preset H-Score score distinguishes between high and low expression of the OARD1 protein. A prognostic prediction model is constructed by combining the subject's TNM stage to assess the risk of disease progression in breast cancer patients.

[0035] In this invention, the provided OARD1 protein can be used for breast cancer diagnosis or prognosis. According to the experiments of this invention, the expression of OARD1 protein in breast cancer tissue is significantly higher than that in adjacent normal tissue, and it can be used as a biomarker for breast cancer diagnosis. Moreover, it shows that the expression of OARD1 protein is closely related to the TNM stage and survival of breast cancer patients.

[0036] In some embodiments, when the preset H-Score is 4.5, the diagnostic sensitivity is 93.3% and the specificity is 88.6%.

[0037] In some embodiments, when H-Score > 4.5, the diagnostic accuracy is ≥ 90.48%.

[0038] In some embodiments, when the preset H-Score is a median of 4, the patients are grouped according to their H-Score. Those with scores higher than the median are in the high expression group, and those with scores lower than or equal to the median are in the low expression group. In this case, the prediction formula of the model is: Risk score = 0.38 × protein expression level assignment + 0.62 × TNM stage assignment. When the risk score is ≥2.48, the 5-year survival rate of the patients is ≤48.9%. Among them, the protein expression level assignment is: low expression = 0, high expression = 2; the TNM stage assignment is: stage I = 1, stage II = 2, stage III = 4, stage IV = 6.

[0039] In this invention, the prediction formula is constructed based on 5-year follow-up data of 172 breast cancer patients. The model's AUC value is 0.82, and the 95% confidence interval is 0.75–0.89. The protein expression distribution corresponding to each TNM stage is as follows: Stage I: low protein expression in 24 cases, high expression in 15 cases; Stage II: low protein expression in 34 cases, high expression in 52 cases; Stage III: low protein expression in 6 cases, high expression in 34 cases; Stage IV: low protein expression in 0 cases, high expression in 7 cases.

[0040] In some embodiments, the criteria for determining the H-Score are as follows:

[0041] The H-Score is calculated by combining the staining area score (percentage of positively stained area) and the staining intensity score in immunohistochemistry. The staining area score ranges from 0 to 4: 0 indicates no immunostaining, 1 indicates 1% to 14% positive, 2 indicates 15% to 49% positive, 3 indicates 50% to 74% positive, and 4 indicates 75% or more positive. The staining intensity score ranges from 0 to 4: 0 (no color), 1 (light yellow), 2 (light brown), 3 (brown), and 4 (dark brown). The H-Score for each tissue is calculated by adding the staining intensity score and the staining area score (therefore, the H-Score ranges from 0 to 8).

[0042] According to a third aspect of the present invention, a kit for the diagnosis and / or prognostic assessment of breast cancer is provided, comprising a reagent that specifically binds to the OARD1 protein; the reagent enables the detection of the OARD1 protein expression level in a sample by immunohistochemistry.

[0043] In this invention, the reagents that specifically bind to the OARD1 protein include anti-OARD1 monoclonal antibodies, anti-OARD1 polyclonal antibodies, or anti-OARD1 nanobodies.

[0044] In this invention, immunohistochemical staining was used to detect samples during the experiment, which confirmed that OARD1 protein can be used as a molecular marker for the diagnosis of breast cancer. The method is simple and easy to implement, the diagnostic process is safe and effective, it is easily accepted by patients, the diagnostic criteria are uniform, and it is less affected by personal subjective factors. This further demonstrates that OARD1 protein is of great significance for the prognosis of breast cancer.

[0045] In some embodiments, the kit further includes antigen retrieval solution, HRP-labeled secondary antibody, DAB chromogenic solution, positive control, and negative control.

[0046] In some embodiments, the conditions for immunohistochemical staining include: primary antibody incubation temperature of 2°C to 10°C and incubation time of 1 to 16 hours.

[0047] In some embodiments, the conditions for immunohistochemical staining include: secondary antibody incubation at room temperature for 10-60 minutes.

[0048] In some embodiments, the positive control is a recombinant plasmid expression product containing OARD1 protein (concentration 50 ng / μL), and the negative control is a lysate of normal breast tissue without OARD1 protein; the antigen retrieval solution is a citrate buffer at pH 6.0, with the addition of 0.05% Triton X-100, which can increase the antigen exposure efficiency by more than 30%.

[0049] According to a fourth aspect of the present invention, a breast cancer diagnosis and / or prognostic assessment system based on OARD1 protein expression levels is provided, comprising:

[0050] The data acquisition module is used to acquire OARD1 protein-related data of subjects or to acquire OARD1 protein-related data and TNM staging information of patients; the OARD1 protein-related data is the H-Score score of OARD1 protein or OARD1 protein expression level data;

[0051] The analysis and processing module, which is communicatively connected to the data acquisition module, is used to perform at least one of the following operations:

[0052] (1) Diagnostic analysis: The H-Score of the OARD1 protein is compared with the positive diagnostic threshold for breast cancer described in this invention to generate a positive or negative diagnostic indicator for breast cancer;

[0053] (2) Prognostic analysis: Input the OARD1 protein expression level data and TNM staging information into the prognostic prediction model of the present invention to generate a prognostic indicator of the patient's 5-year survival risk;

[0054] The result output module is communicatively connected to the analysis and processing module and is used to output the diagnostic indication or prognosis indication.

[0055] In some embodiments, the data acquisition module includes an immunohistochemical detection data interface for receiving the OARD1 protein H-Score obtained after immunohistochemical detection of breast tissue or breast cancer tissue samples.

[0056] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0057] Unless otherwise specified, the raw materials used in the following embodiments are all disclosed in the prior art, such as those that can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0058] Example 1

[0059] 1. Source of Tissue Samples: Samples from 172 breast cancer patients were obtained from the tissue sample bank of Shanghai Zhuohao Pharmaceutical Technology Co., Ltd. The surgical period was from January 2004 to August 2017, and the final follow-up date was April 2022. All cases were pathologically confirmed as breast cancer, and all patients were female, aged 31-84 years, with a median age of 48 years. Of these 172 samples, 130 contained both cancerous tissue and matching adjacent tissue within 1.5 cm of the cancer, while the remaining 42 contained only cancerous tissue. The case data of the 172 patients are shown in Table 1.

[0060] Table 1. Clinicopathological data of 172 breast cancer patients

[0061]

[0062] 2. The chip fabrication process is as follows:

[0063] The tissue microarrays were fabricated by Shanghai Zhuohao Pharmaceutical Technology Co., Ltd. All donor tissue blocks underwent routine pathological sectioning and HE staining, followed by secondary diagnosis by pathologists, who marked typical pathological sites on the HE sections. Using a Beecher Instruments Inc. tissue microarray fabrication system, 1.5 mm diameter holes were drilled in blank recipient tissue blocks. Target tissue cores were then obtained from the corresponding locations on the donor tissue blocks according to the markings on the HE sections and placed into the array wells of the recipient blocks. This process was repeated to create the BRC1601 and BRC1603 dot arrays matching breast cancer tissue and adjacent normal tissue. Sections were continuously sliced ​​at a thickness of 4 μm using a Leica (Germany) microtome, baked at 65°C for 2 hours, dewaxed, and mounted on imported glass slides treated to prevent detachment.

[0064] 3. Experimental Methods

[0065] Immunohistochemical experiments were performed on the prepared tissue microarrays using a two-step immunohistochemistry kit from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0066] (1) Dewaxing of breast cancer tissue microarray: Place the breast cancer tissue microarray on a baking machine and bake at 65°C for two hours. Then, dewax the tissue microarray in a fume hood.

[0067] (2) After dewaxing, the breast cancer tissue chip was rinsed in tap water for 5 minutes; then it was placed in an immunohistochemistry box containing ddH2O and washed on a shaker for 5 minutes.

[0068] (3) Antigen retrieval: Add antigen retrieval solution to the immunohistochemistry box, and then place the breast cancer tissue chip in it; adjust the temperature of the constant temperature water bath to 99°C, put the immunohistochemistry box containing the breast cancer tissue chip into the water bath, and use a thermometer to measure the real-time temperature in the immunohistochemistry box. When the temperature rises to 95°C, start timing and antigen retrieval for 18 minutes.

[0069] (4) After antigen retrieval is completed, remove the immunohistochemistry box from the water bath and allow the breast cancer tissue chip to return to room temperature.

[0070] (5) After the breast cancer tissue microarray has returned to room temperature, PBST solution is added to the immunohistochemistry box, the breast cancer tissue microarray is placed in it, and it is washed three times on a shaker for 5 minutes each time.

[0071] (6) Removal of peroxidase: Remove the breast cancer tissue chip from the immunohistochemistry box and wipe the liquid around the tissue with absorbent paper; draw a circle around the tissue with an immunohistochemistry pen to enclose the tissue in the circle; then place the breast cancer tissue chip in a humidified box, add 3% hydrogen peroxide solution to the tissue, and incubate in the dark for 10 min.

[0072] (7) After the peroxidase removal is completed, PBST solution is added to the immunohistochemistry box, the breast cancer tissue chip is placed in it, and it is washed three times on a shaker for 10 minutes each time.

[0073] (8) Blocking: Remove the breast cancer tissue chip from the immunohistochemistry box, shake off the liquid on the slide, place the breast cancer tissue chip in a humidified box, add blocking solution to the tissue, cover the humidified box, and block at room temperature for one and a half hours.

[0074] (9) Preparation of primary antibody: Prepare OARD1 primary antibody (Invitrogen / catalog number PA5-148364) with antibody diluent at a ratio of 1:200.

[0075] (10) Incubation of primary antibody: After the blocking is completed, the blocking solution on the tissue is removed, the breast cancer tissue chip is placed in a humidified box, the freshly prepared primary antibody solution is added to the tissue, the lid is closed, and it is incubated overnight in a refrigerator at 4°C.

[0076] (11) After the primary antibody incubation is completed, remove the wet box from the 4°C refrigerator and place it at room temperature for half an hour to allow the breast cancer tissue chip to return to room temperature.

[0077] (12) After the temperature returns to room temperature, add PBST solution to the immunohistochemistry box, place the breast cancer tissue chip in it, and wash it three times on a shaker for 10 minutes each time.

[0078] (13) Incubation of secondary antibody: Remove the PBST liquid from the tissue, place the breast cancer tissue chip in a humidified chamber, add HRP-labeled goat anti-rabbit secondary antibody to the tissue, cover the chamber, and incubate at room temperature for one hour.

[0079] (14) After the secondary antibody incubation is completed, add PBST solution to the immunohistochemistry box, put the breast cancer tissue chip into it, and wash it three times on a shaker for 10 minutes each time.

[0080] (15) Preparation of DAB colorimetric solution: Prepare DAB colorimetric solution at a ratio of 1:20, use in the dark, and prepare and use immediately.

[0081] (16) DAB staining: Shake off the PBST liquid on the tissue, place the breast cancer tissue chip under a microscope, add DAB staining solution to the tissue for staining, and when the specific staining is obvious and the background is clean, place the breast cancer tissue chip in tap water to stop the staining.

[0082] (17) Hematoxylin staining: The breast cancer tissue chip was stained in hematoxylin solution for 2 min 30 s and then rinsed in tap water for 5 min.

[0083] (18) Hydrochloric acid alcohol differentiation: Dip the breast cancer tissue chip into hydrochloric acid alcohol, remove it quickly, and rinse it in tap water for 5 minutes.

[0084] (19) Blueing with dilute ammonia: Place the breast cancer tissue chip in dilute ammonia for 1 min 30 s to blue, rinse in tap water for 5 min, and dehydrate the tissue.

[0085] (20) Tissue mounting: After the breast cancer tissue microarray is dehydrated, the slide is removed from the clearing solution, neutral resin is dropped onto the tissue, and then a coverslip is placed on top.

[0086] 4. Quantitative analysis is as follows:

[0087] The staining results were photographed, and the positive area and staining intensity within each section were converted into corresponding H-Score values ​​to achieve semi-quantitative analysis of tissue staining. The histochemical H-Score ranged from a minimum of 0 to a maximum of 8, with a median of 4. Patients were grouped according to their H-Score: those above the median were classified as high-expression, and those below or equal to the median were classified as low-expression.

[0088] 5. Statistical analysis is as follows:

[0089] The expression of OARD1 protein in breast cancer and adjacent normal tissues was analyzed using a t-test. The correlation between OARD1 protein expression and clinical indicators in breast cancer patients was analyzed using a chi-square test. The correlation between OARD1 protein and the prognosis of breast cancer patients was analyzed using Kaplan-Meier survival analysis and log-rank statistical tests for univariate survival. A p-value < 0.05 was considered statistically significant.

[0090] 6. The experimental results are as follows:

[0091] (1) Expression analysis of OARD1 protein in breast cancer and adjacent normal tissues.

[0092] Immunohistochemical analysis results as follows Figure 1 As shown, the expression of OARD1 protein in breast cancer tissue was significantly higher than that in adjacent normal tissue (p < 0.001).

[0093] (2) Correlation between OARD1 protein and clinical indicators in breast cancer patients.

[0094] Breast cancer patients were grouped according to factors such as age or TNM stage, and the correlation between different factors and OARD1 protein expression was analyzed using the chi-square test. The results are shown in Table 2. The expression level of OARD1 protein in breast cancer was not significantly related to the patient's age (p>0.05), but was closely related to the patient's TNM stage (p<0.001).

[0095] Table 2. Correlation between OARD1 protein expression and clinical indicators in breast cancer patients.

[0096]

[0097] (3) Follow-up of breast cancer patients is as follows: the surgery period was from January 2004 to August 2017, and the final follow-up period was April 2022. Univariate analysis of survival using Kalplan-Meier survival analysis and log-rank statistical test showed that, as Figure 2As shown, breast cancer patients with low OARD1 expression in their cancerous tissue had a longer overall survival (p=0.0002), and their 5-year survival rate was significantly higher than that of breast cancer patients with high OARD1 expression. This result suggests that OARD1 detection is of significant importance for prognostic assessment of breast cancer.

[0098] Example 2

[0099] S1. Source of tissue samples, as follows:

[0100] The cancer tissue samples from 105 breast cancer patients and the adjacent normal tissue samples from 105 breast cancer patients were obtained from the tissue sample bank of Shanghai Xinchao Biotechnology Co., Ltd.

[0101] S2. Fabrication of tissue microarrays, the method is as follows:

[0102] The tissue microarrays were fabricated by Shanghai Chipover Biotechnology Co., Ltd. Using the TMA Grand Master tissue microarray fabrication instrument, target tissue cores were obtained by drilling holes in the recipient paraffin blocks and placed into the array wells of the recipient paraffin blocks. The hole diameter was 1.5 mm. This process was repeated to finally fabricate the dot array blocks HBreD120CS01 and HBreD090CS01 of breast cancer tissue and adjacent normal tissue. Paraffin sections were continuously sectioned at a thickness of 4 μm using a Leica paraffin microtome (Germany). After baking at 65°C for 2 hours, the sections were dewaxed and mounted on imported glass slides treated to prevent detachment.

[0103] 3. Immunohistochemical staining experiment, the method is as follows:

[0104] Immunohistochemical experiments were performed on the prepared tissue microarrays using a two-step immunohistochemistry kit from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0105] (1) Dewaxing of breast cancer tissue microarray: Place the breast cancer tissue microarray on a baking machine and bake at 65°C for two hours. Then, dewax the tissue microarray in a fume hood.

[0106] (2) After dewaxing, the breast cancer tissue chip was rinsed in tap water for 5 minutes; then it was placed in an immunohistochemistry box containing ddH2O and washed on a shaker for 5 minutes.

[0107] (3) Antigen retrieval: Add antigen retrieval solution to the immunohistochemistry box, and then place the breast cancer tissue chip in it; adjust the temperature of the constant temperature water bath to 99°C, put the immunohistochemistry box containing the breast cancer tissue chip into the water bath, and use a thermometer to measure the real-time temperature in the immunohistochemistry box. When the temperature rises to 95°C, start timing and antigen retrieval for 18 minutes.

[0108] (4) After antigen retrieval is completed, remove the immunohistochemistry box from the water bath and allow the breast cancer tissue chip to return to room temperature.

[0109] (5) After the breast cancer tissue microarray has returned to room temperature, PBST solution is added to the immunohistochemistry box, the breast cancer tissue microarray is placed in it, and it is washed three times on a shaker for 5 minutes each time.

[0110] (6) Removal of peroxidase: Remove the breast cancer tissue chip from the immunohistochemistry box and wipe the liquid around the tissue with absorbent paper; draw a circle around the tissue with an immunohistochemistry pen to enclose the tissue in the circle; then place the breast cancer tissue chip in a humidified box, add 3% hydrogen peroxide solution to the tissue, and incubate in the dark for 10 min.

[0111] (7) After the peroxidase removal is completed, PBST solution is added to the immunohistochemistry box, the breast cancer tissue chip is placed in it, and it is washed three times on a shaker for 10 minutes each time.

[0112] (8) Blocking: Remove the breast cancer tissue chip from the immunohistochemistry box, shake off the liquid on the slide, place the breast cancer tissue chip in a humidified box, add blocking solution to the tissue, cover the humidified box, and block at room temperature for one and a half hours.

[0113] (9) Preparation of primary antibody: Prepare OARD1 primary antibody with antibody dilution solution at a ratio of 1:200.

[0114] (10) Incubation of primary antibody: After the blocking is completed, the blocking solution on the tissue is removed, the breast cancer tissue chip is placed in a humidified box, the freshly prepared primary antibody solution is added to the tissue, the lid is closed, and it is incubated overnight in a refrigerator at 4°C.

[0115] (11) After the primary antibody incubation is completed, remove the wet box from the 4°C refrigerator and place it at room temperature for half an hour to allow the breast cancer tissue chip to return to room temperature.

[0116] (12) After the temperature returns to room temperature, add PBST solution to the immunohistochemistry box, place the breast cancer tissue chip in it, and wash it three times on a shaker for 10 minutes each time.

[0117] (13) Incubation of secondary antibody: Remove the PBST liquid from the tissue, place the breast cancer tissue chip in a humidified chamber, add HRP-labeled goat anti-rabbit secondary antibody to the tissue, cover the chamber, and incubate at room temperature for one hour.

[0118] (14) After the secondary antibody incubation is completed, add PBST solution to the immunohistochemistry box, put the breast cancer tissue chip into it, and wash it three times on a shaker for 10 minutes each time.

[0119] (15) Preparation of DAB colorimetric solution: Prepare DAB colorimetric solution at a ratio of 1:20, use in the dark, and prepare and use immediately.

[0120] (16) DAB staining: Shake off the PBST liquid on the tissue, place the breast cancer tissue chip under a microscope, add DAB staining solution to the tissue for staining, and when the specific staining is obvious and the background is clean, place the breast cancer tissue chip in tap water to stop the staining.

[0121] (17) Hematoxylin staining: The breast cancer tissue chip was stained in hematoxylin solution for 2 min 30 s and then rinsed in tap water for 5 min.

[0122] (18) Hydrochloric acid alcohol differentiation: Dip the breast cancer tissue chip into hydrochloric acid alcohol, remove it quickly, and rinse it in tap water for 5 minutes.

[0123] (19) Blueing with dilute ammonia: Immerse the breast cancer tissue chip in dilute ammonia for 1 min 30 s to achieve blueing, rinse in tap water for 5 min, and dehydrate the tissue.

[0124] (20) Tissue mounting: After the breast cancer tissue microarray is dehydrated, the slide is removed from the clearing solution, neutral resin is dropped onto the tissue, and then a coverslip is placed on top.

[0125] 4. Quantitative analysis, the methods are as follows:

[0126] The staining results were photographed, and the positive area and staining intensity within each section were converted into corresponding H-Score values ​​to achieve semi-quantitative analysis of tissue staining. The histochemical H-Score ranged from a minimum of 0 to a maximum of 8, with a median of 4. Patients were grouped according to their H-Score: those above the median were classified as high-expression, and those below or equal to the median were classified as low-expression.

[0127] 5. Statistical analysis is as follows:

[0128] The diagnostic efficacy of OARD1 protein expression intensity for breast cancer was analyzed using ROC curve analysis. AUC, the area under the ROC curve, is the most commonly used parameter for evaluating the characteristics of the ROC curve and an important indicator of experimental accuracy.

[0129] 6. The experimental results are as follows:

[0130] like Figure 3 As shown, the area under the ROC curve (AUC) in this invention was 0.948, with a 95% confidence interval of 0.917–0.978 (p < 0.001), indicating that OARD1 protein can serve as a biomarker for diagnosing breast cancer. When the immunohistochemical score (H-Score) of OARD1 protein was 4.5, the sensitivity was 93.3% and the specificity was 88.6%. In individual testing, an H-Score greater than 4.5 for OARD1 protein was considered indicative of breast cancer, with an accuracy of 90.48%.

[0131] Example 3

[0132] 1. Sample source: Cancer tissue samples from 172 breast cancer patients in Example 1;

[0133] 2. Experimental methods: A prognostic prediction model was constructed by combining clinical indicators such as the expression level of OARD1 protein, TNM stage of patients, and follow-up survival status in Table 3.

[0134] Table 3

[0135]

[0136] 3. Experimental Results:

[0137] The prognostic prediction model uses the following formula: Risk Score = 0.38 × Protein Expression Level Assignment + 0.62 × TNM Stage Assignment. When the risk score is ≥2.48, the 5-year survival rate is ≤48.9%. Protein expression level assignments are: low expression = 0, high expression = 2; TNM stage assignments are: Stage I = 1, Stage II = 2, Stage III = 4, Stage IV = 6. The model's AUC value is 0.82, significantly higher than using TNM staging alone (AUC = 0.786).

[0138] Risk scoring formula (adjusted coefficient logic + Cox regression validation + ROC efficacy validation): Based on updated 5-year follow-up data of 172 breast cancer patients, a Cox proportional hazards regression model was used for multivariate analysis to validate and determine the weight coefficients of each variable. "Protein expression level" (low expression = 0, high expression = 2) and "TNM stage" (Stage I = 1, Stage II = 2, Stage III = 4, Stage IV = 6) were used as independent variables, and "5-year survival status" (survival = 0, death = 1) was used as the dependent variable. Regression analysis showed that: ① High OARD1 protein expression is a unique factor contributing to poor prognosis in breast cancer. ① Risk factors (HR=1.86, 95%CI: 1.12~3.08, P=0.016); ② Elevated TNM stage was an independent risk factor for poor prognosis (Stage I was the reference; Stage II HR=1.92, 95%CI: 0.75~4.93, P=0.178; Stage III HR=4.25, 95%CI: 1.73~10.47, P=0.002; Stage IV HR=9.63, 95%CI: 3.85~24.09, P<0.001); ③ Based on the standardized regression coefficient results, the final formula was determined as: Risk score = 0.38 × protein expression level assignment + 0.62 × TNM stage assignment. This coefficient setting retains the dominant role of TNM stage (a core traditional prognostic indicator) while strengthening the independent predictive value of OARD1 protein expression. Furthermore, it was verified by the Hosmer-Lemeshow test for goodness of fit. =6.32, P=0.612) indicates a good model fit (the null hypothesis of the Hosmer-Lemeshow test is "good model fit," and if P>0.05, the null hypothesis is not rejected, indicating that there is no significant difference between the model's predicted value and the actual survival outcome, i.e., the model fit is good; if P<0.05, the null hypothesis is rejected, indicating a poor model fit). Further verification of the model's predictive efficacy was achieved through ROC curve analysis: using "5-year death" as the outcome event and the risk score as a predictor variable, an ROC curve was plotted. The results showed that the area under the curve (AUC) = 0.82 (95% confidence interval: 0.75~0.89, P<0.001), indicating that the risk score model has good predictive efficacy for the 5-year survival outcome of breast cancer patients (AUC≥0.8 indicates excellent predictive efficacy).

[0139] Threshold determination: A score ≥ 2.48 was used as the high-risk threshold, corresponding to a group of 47 people with low expression in stage III (6 people) + high expression in stage III (34 people) + high expression in stage IV (7 people), with 24 deaths and a survival rate of (47-24) / 47≈48.9%; A score ≥ 2.86 was used as the very high-risk threshold, corresponding to a group of 41 people with high expression in stage III (34 people) + high expression in stage IV (7 people), with 23 deaths and a survival rate of ≈43.9%; A score ≥ 4.06 was used as the extremely high-risk threshold, corresponding to a group of 7 people with high expression in stage IV, with a survival rate of 0.0%.

[0140] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. Use of an agent that specifically binds to an OARD1 protein for the manufacture of a diagnostic product for breast cancer, characterized in that, The product detects the expression level of OARD1 protein in the breast tissue sample by a reagent specifically binding to OARD1 protein and performs H-Score scoring, takes a preset H-Score score as a breast cancer positive diagnosis threshold, and judges whether the sample is breast cancer positive.

2. The use according to claim 1, characterized in that, when the preset H-Score score is 4.5, the diagnostic sensitivity is 93.3%, the specificity is 88.6%; and / or when the H-Score is > 4.5, the diagnostic accuracy is ≥ 90.48%.

3. Use of an agent that specifically binds to an OARD1 protein for the manufacture of a breast cancer prognosis evaluation product, characterized in that, The product detects the expression level of OARD1 protein in the breast cancer tissue sample by a reagent specifically binding to OARD1 protein and performs H-Score scoring, distinguishes high / low expression of OARD1 protein by a preset H-Score score, constructs a prognosis prediction model combined with the TNM stage of the subject, and realizes the evaluation of the disease progression risk of the breast cancer patient.

4. The use according to claim 3, characterized in that, when the preset H-Score score is the median value 4, the H-Score score is used for grouping, the high expression group is higher than the median value, and the low expression group is lower than or equal to the median value, at this time, the prediction formula of the model is: risk score = 0.38 x protein expression level assignment + 0.62 x TNM stage assignment, when the risk score is ≥ 2.48, the 5-year survival rate of the patient is ≤ 48.9%; wherein the protein expression level assignment is: low expression = 0, high expression = 2; the TNM stage assignment is: stage I = 1, stage II = 2, stage III = 4, stage IV = 6.

5. Use according to claim 1 or 3, characterized in that, The determination criteria of the H-Score score are: The H-Score value is calculated by adding the staining area score and the staining intensity score in the immunohistochemical method; wherein the staining area score ranges from 0 to 4: 0 represents no immunostaining, 1 represents 1% to 14% of the area being positive, 2 represents 15% to 49% of the area being positive, 3 represents 50% to 74% of the area being positive, and 4 represents 75% and above of the area being positive; the staining intensity score ranges from 0 to 4: 0 represents no color, 1 represents light yellow, 2 represents light brown, 3 represents brown, and 4 represents dark brown.

6. A breast cancer diagnosis and / or prognosis evaluation system based on the expression level of OARD1 protein, characterized in that, It comprises: a data acquisition module for acquiring OARD1 protein related data of a subject or for acquiring OARD1 protein related data of a patient and TNM stage information; the OARD1 protein related data is H-Score score of OARD1 protein or OARD1 protein expression level data; an analysis processing module in communication connection with the data acquisition module, configured to perform at least one of the following operations: (1) diagnostic analysis: comparing the H-Score score of the OARD1 protein with a breast cancer positive diagnosis threshold to generate a diagnosis indication of breast cancer positive or negative; (2) prognosis analysis: inputting the OARD1 protein expression level data and the TNM stage information into a prognosis prediction model to generate a prognosis indication of the 5-year survival risk of the patient; An output module, in communication with the analysis processing module, for outputting the diagnostic or prognostic indication.

7. The system of claim 6, wherein, The data acquisition module comprises an immunohistochemical detection data interface for receiving an OARD1 protein H-Score score obtained after detecting a breast tissue or breast cancer tissue sample by an immunohistochemical method.

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