Use of a multiprotein marker and kit and system for early diagnosis of breast cancer

Abstract

The application discloses a kind of application of multi-protein marker and kit and system for early diagnosis of breast cancer, belong to biomarker technical field.The multi-protein marker includes PTN, IL12, CCL3, CASP-8 and ADGRG1.The application first five proteins PTN, IL12, CCL3, CASP-8 and ADGRG1 are used as combination marker for early diagnosis of breast cancer, and corresponding ELISA kit, diagnostic system and judging method are constructed, and the diagnostic method is high in sensitivity and specificity, can improve the early detection rate of breast cancer while guaranteeing specificity, and the early screening kit and diagnostic system constructed can also be applied to the screening of therapeutic drug for breast cancer, has wide application prospect, solves the problem of insufficient sensitivity, low specificity and poor stability of single index in the existing early diagnosis of breast cancer serology marker.

Description

Technical Field

[0001] This invention relates to the field of biomarker technology, specifically to the application of a multi-protein biomarker and a kit and system for the early diagnosis of breast cancer. Background Technology

[0002] Breast cancer is one of the most common malignant tumors among women worldwide. In my country, the incidence of breast cancer has been rising steadily in recent years, becoming the most common malignant tumor among women. Clinical data shows that early diagnosis is crucial for improving prognosis: the 5-year survival rate for stage I breast cancer patients can exceed 90%, while that for stage IV patients is less than 30%. However, existing screening strategies still have limitations, such as limited sensitivity and a high false negative rate, making it difficult to meet the clinical needs for early and accurate diagnosis.

[0003] Currently, early clinical screening for breast cancer mainly relies on imaging methods such as mammography, ultrasound, and MRI. However, these methods have low sensitivity for identifying early lesions in women with dense breasts. Traditional serum tumor markers (CA15-3, CEA) lack sufficient sensitivity and specificity in early breast cancer, making it difficult to meet the needs of early clinical screening. Therefore, exploring sensitive, specific, reproducible, and scalable molecular diagnostic markers has become an important direction for breast cancer prevention and control.

[0004] Against this backdrop, non-invasive liquid biopsy technology is becoming a frontier in global early cancer screening research due to its advantages such as not requiring tissue biopsy, ease of repeated testing, and ability to reflect dynamic changes in tumors. Among them, the Olink high-throughput proteomics platform based on the proximity extension assay (PEA) has outstanding advantages in breast cancer liquid biopsy research due to its ultra-high sensitivity, low sample volume requirements, and ability to detect multiple indicators simultaneously.

[0005] However, the stability and accuracy of single-protein biomarkers in the early diagnosis of breast cancer remain limited, necessitating the development of multi-protein combined detection schemes to improve diagnostic performance and enhance clinical applicability. Therefore, developing a method and kit for the early diagnosis of breast cancer based on multi-protein combined detection has significant clinical value and application prospects. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide an application of multi-protein biomarkers and a kit and system for the early diagnosis of breast cancer, thereby solving the problems of insufficient sensitivity, low specificity, and poor stability of single indicators in existing early diagnosis of breast cancer serological biomarkers.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides the use of multi-protein biomarkers in the preparation of breast cancer therapeutic agents or early screening kits, the multi-protein biomarkers including pleiotropic neurotrophic factor (PTN), interleukin-12 (IL12), macrophage inflammatory protein 1-α (CCL3), caspase-8 (CASP-8), and adhesion G protein-coupled receptor G1 (ADGRG1).

[0008] The beneficial effects of this invention are as follows: This invention is the first to use five proteins, PTN, IL12, CCL3, CASP-8 and ADGRG1, as combined biomarkers for the early diagnosis of breast cancer, and constructs corresponding ELISA kits, diagnostic systems and judgment methods. The diagnostic methods have high sensitivity and specificity, and can improve the early detection rate of breast cancer while ensuring specificity. At the same time, the constructed early screening kits and diagnostic systems can also be applied to the screening of therapeutic drugs for breast cancer, and have broad application prospects.

[0009] Furthermore, among the multi-protein biomarkers, pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, and adhesion G protein-coupled receptor G1 were upregulated in the plasma of breast cancer patients, while caspase-8 was downregulated in the plasma of breast cancer patients.

[0010] In a second aspect, the present invention provides an early breast cancer screening kit, comprising a capture antibody component, a detection antibody component, a standard component, auxiliary reagents, and an interpretation module; The capture antibody component includes specific capture antibodies against pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, caspase 8, and adhesion G protein-coupled receptor G1, respectively. The detection antibody component includes corresponding detection antibodies for pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, caspase 8, and adhesion G protein-coupled receptor G1. The standard components include pleiotropic neurotrophic factor at known concentration gradients, interleukin-12, macrophage inflammatory protein 1-α, caspase 8, and adhesion G protein-coupled receptor G1 standard protein.

[0011] The beneficial effects of this invention are as follows: The kit provided by this invention can be applied to early rapid screening of breast cancer, auxiliary diagnosis of people with uncertain imaging examination results, and preoperative and postoperative efficacy monitoring and risk assessment of breast cancer patients. Based on the ELISA platform, it is easy to operate, low in cost, and has good reproducibility, making it suitable for physical examinations and primary hospitals, and making up for the shortcomings of imaging in early small lesions.

[0012] Furthermore, auxiliary reagents include blocking solution, washing solution, chromogenic substrate, stop solution, and reaction buffer.

[0013] Furthermore, the interpretation module includes instructions or software that converts the test results into diagnostic scores and provides reference thresholds.

[0014] Furthermore, the kit is a 96-well plate structure, which can enable the simultaneous or parallel detection of five proteins.

[0015] Furthermore, the antibodies are labeled with HRP or AP, and the OD value is read by colorimetry and converted into concentration.

[0016] A third aspect of the present invention provides a method for establishing an early breast cancer screening system, comprising the following steps: S1. Collect peripheral blood samples from breast cancer patients and normal individuals, and separate plasma or serum as the test samples; S2. The expression levels of pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, caspase 8 and adhesion G protein-coupled receptor G1 in the test sample obtained in S1 were detected using the above-mentioned kit, and the detection results were obtained. S3. Construct a joint diagnostic model based on the detection results obtained in S3, and determine the diagnostic cutoff value.

[0017] Furthermore, the joint diagnostic model in S3 is a linear or nonlinear regression model, including the following forms: Score=α1×PTN+α2×IL12+α3×CCL3+α4×CASP-8+α5×ADGRG1+β; Among them, α1-α5 are the weight coefficients of pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, caspase 8 and adhesion G protein-coupled receptor G1, respectively. PTN, IL12, CCL3, CASP-8, and ADGRG1 are the detection results of pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, caspase 8, and adhesion G protein-coupled receptor G1, respectively. β is a constant term; Score is the diagnostic score; When the diagnostic score is greater than or equal to the diagnostic cutoff value, the subject is determined to be a high-risk individual for breast cancer; When the diagnostic score is less than the diagnostic cutoff value, the subject is considered a low-risk individual for breast cancer.

[0018] Furthermore, the diagnostic cutoff values ​​are determined based on training samples using statistical or machine learning methods, including: 1) The point where the Youden exponent is at its maximum on the ROC curve; 2) The dividing point under preset sensitivity or specificity conditions; 3) The optimal threshold obtained through cross-validation or independent validation arrays.

[0019] In a fourth aspect, the present invention provides an early breast cancer screening system obtained by the above-described method.

[0020] A fifth aspect of the present invention provides a method for early diagnosis of breast cancer based on an early breast cancer screening system: (1) Sample acquisition Peripheral blood samples were collected from the subjects, and plasma or serum was separated as the test samples. (2) Protein detection The expression levels of pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, caspase 8 and adhesion G protein-coupled receptor G1 were detected using the above kit. (3) Data processing The detection concentration values ​​or standardized expression levels of the five proteins are input into the early breast cancer screening system to obtain a diagnostic score; (4) Result determination The diagnostic score was compared with the diagnostic cutoff value. When the diagnostic score was greater than or equal to the diagnostic cutoff value, the subject was determined to be a high-risk individual for breast cancer. When the diagnostic score is less than the diagnostic cutoff value, the subject is considered a low-risk individual for breast cancer.

[0021] The present invention has the following beneficial effects: 1. This invention uses a combination of five proteins—PTN, IL12, CCL3, CASP-8, and ADGRG1—for the early diagnosis of breast cancer. Among the single proteins, PTN has the best diagnostic performance. IL12, CCL3, CASP-8, and ADGRG1 also have certain discriminative power. The AUC of the constructed combined model is significantly higher than any single indicator, which significantly improves sensitivity and specificity.

[0022] 2. The multi-protein biomarker provided by this invention uses PTN as the core. PTN is significantly elevated in preoperative samples and significantly decreased after surgery, indicating that it is highly correlated with tumor burden. Using PTN as the core weight and combining it with the other four proteins can improve the early detection rate while ensuring specificity.

[0023] 3. This invention constructs a multi-protein biomarker early diagnostic kit for breast cancer based on the ELISA platform. It is easy to operate, low in cost, and has good reproducibility, making it suitable for physical examinations and primary hospitals, thus compensating for the shortcomings of imaging in the early detection of small lesions.

[0024] 4. This invention is the first to combine five proteins, PTN, IL12, CCL3, CASP-8 and ADGRG1, for the early diagnosis of breast cancer, and constructs corresponding ELISA kits, diagnostic models and skewness methods, which have clear inventiveness and industrial value. Attached Figure Description

[0025] Figure 1 This is a flowchart of the Olink high-throughput detection process. Figure 2 This is an analysis of differentially expressed proteins in preoperative plasma of breast cancer patients and healthy controls in Example 1. In this example, A is a hierarchical clustering heatmap of differentially expressed proteins, showing the difference in expression patterns between preoperative breast cancer samples and healthy controls; B is a volcano plot showing the differentially expressed proteins; and C is a box plot showing the expression distribution of differentially expressed proteins in the two groups. Figure 3 The ROC curve and multi-index joint diagnostic model performance analysis in Example 1 are shown, where A is the ROC curve of a single index and B is the ROC curve of multiple indices combined. Figure 4 Example 2 shows the analysis of differentially expressed proteins in the plasma of breast cancer patients before and after surgery. In this example, A is a hierarchical clustering heatmap of differentially expressed proteins, showing the difference in expression patterns between preoperative breast cancer samples and healthy controls; B is a volcano plot showing the differentially expressed proteins; and C is a box plot showing the expression distribution of differentially expressed proteins in the two groups. Figure 5 The results of ELISA detection of PTN in plasma in Example 3 are shown. A is a comparison of PTN expression levels in healthy controls (NC), pre-operative (PRE), and post-operative (POST) samples of breast cancer, and B is a comparison of PTN expression changes in paired samples of the same patient before and after surgery. Figure 6 The ROC curve for PTN single index in Example 3; Figure 7 The image shows the immunohistochemical staining results of PTN in breast cancer tissue in Example 4, where A represents the staining results and B is a statistical graph. Detailed Implementation

[0026] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0027] Example 1: Screening and Determination of Multiprotein Biomarkers I. Sample Source Thirty-one breast cancer patients admitted to the Cancer Hospital of Ningxia Medical University General Hospital from January to July 2025 were included in this study, along with 20 healthy female controls, all age-matched. Approximately 2 mL of whole blood samples were collected from patients before surgery and 3-4 weeks after treatment. The samples were centrifuged at 2000 g, 4°C for 10 min, and the supernatant was collected, placed in centrifuge tubes, and stored at -80°C for later use.

[0028] (1) Sample inclusion criteria ①Female, 18-75 years old; ②The diagnosis was confirmed by histopathology as non-specific invasive breast cancer; ③Surgical treatment is planned and the first blood sample can be collected before the operation; ④ Follow-up blood collection can be conducted 3-4 weeks after the operation; ⑤ Complete clinical data (including staging, subtype, ER / PR / HER2, Ki-67, treatment information, etc.); ⑥ Sign a written informed consent form.

[0029] (2) Sample discharge standard ① Previous or concurrent primary malignant tumors; ② Acute inflammation / infection (fever, significantly elevated CRP / white blood cell count) within 2 weeks prior to blood collection; ③ Adjustment of the starting dose for those who have received systemic glucocorticoids, immunosuppressants, or immuno / targeted therapy within the past 30 days; ④ During pregnancy or lactation; ⑤ Severe liver and kidney dysfunction, active autoimmune diseases, or hematological diseases; ⑥ There have been events within the past 14 days that significantly affect inflammatory factors, such as major surgery, blood transfusion, or vaccination; ⑦ Inability to cooperate with follow-up or incomplete data. All data collection was ethically approved and informed consent was obtained from the participants.

[0030] II. High-throughput detection This invention utilizes the Olink Target 96 Immune-Oncology Panel to perform multi-target protein quantification on plasma samples from breast cancer patients, and performs quality control and standardization on the obtained protein expression profiles. A schematic diagram of the detection process is shown below. Figure 1 As shown.

[0031] Specifically, the following steps are included: (1) Protein sample preparation; (2) Immunolinking of designed oligonucleotide sequence antibodies with proteins; (3) After the two oligonucleotide sequences attached to the protein undergo base pairing, the paired oligonucleotide sequences are extended in the reaction system under the action of DNA polymerase. (4) Prepare sequence libraries by extending the barcode sequences containing sequencing adapters, sample IDs, and protein IDs; (5) The amplified sequence is then subjected to qPCR detection or NGS sequencing; (6) Convert the bcl file generated by qPCR detection or NGS sequencing into a counts file to obtain the data input file required by NPXSignature software; (7) NPX Signature software data preprocessing; (8) Obtain the input files required for the bioinformatics analysis process: Perform data quality control on the NPX output of NPX Data, including: removing proteins with low detection background signal and excessive deletion; normalization (log2NPX conversion); batch correction (such as ComBat) to eliminate platform and technical variations.

[0032] III. Difference Analysis (1) The preoperative breast cancer samples and healthy controls were compared and the differential expression was analyzed.

[0033] Statistical methods include: ① Normal distribution → t-test; Non-normal distribution → Wilcoxon rank-sum test; ② Multiple false positive control: Benjamini-Hochberg FDR correction; ③ Significance screening criteria: FDR < 0.05 and |log2FC| ≥ 1.

[0034] (2) Results of difference analysis The results of the difference analysis are as follows Figure 2 As shown, seven significantly upregulated proteins (PTN, IL12, IL7, CCL3, ADGRG1, ANGPT1, and CD83) showed an overall increasing trend in the plasma of breast cancer patients, with expression patterns showing strong inter-group differentiation. Among them, PTN showed the most significant change, while CASP-8 was downregulated, suggesting that immune regulation-related proteins have specific changes in the circulating system of breast cancer.

[0035] IV. Determination of Multiprotein Biomarkers Based on statistical significance, AUC values, and biological relevance, the following five proteins were ultimately identified as the diagnostic panel for this invention: PTN, IL12, CCL3, CASP-8, and ADGRG1. The combined model demonstrated significant discriminatory power between preoperative samples and healthy controls, with an AUC > 0.90, an improvement of approximately 0.05-0.10 compared to PTN alone. This indicates that the five-protein combination with PTN as its core possesses excellent clinical diagnostic performance. PTN showed significantly higher levels of differentiation in preoperative breast cancer samples than in healthy controls, and had the highest AUC among the single indicators, thus being identified as the core dominant indicator of this invention. See [link to relevant documentation]. Figure 3 .

[0036] Example 2: Preoperative and postoperative dynamic verification of PTN I. Sample Source Paired plasma samples were selected from 31 breast cancer patients before and after surgery.

[0037] II. High-throughput detection The expression level of PTN was detected using the same quantitative method as in Example 1.

[0038] III. Difference Analysis Experimental results are as follows Figure 4 As shown, the results indicated that MMP12, MCP-4, MCP-3, CXCL11, CASP-8, ARG1, IL6, and CSF-1 were significantly upregulated, while PTN and MMP7 were significantly downregulated. Among these, compared with the previous... Figure 2 In comparison, PTN expression was lower postoperatively than preoperatively, with a statistically significant difference. This indicates that PTN is closely related to tumor burden and has potential application value as a dynamically changing biomarker.

[0039] Example 3: Clinical Validation of ELISA I. Sample Composition The study included 13 preoperative samples from breast cancer patients, 13 postoperative samples from breast cancer patients, and 13 healthy controls.

[0040] II. Experimental Methods This embodiment uses PTN as a representative protein for ELISA detection to verify the clinical feasibility of the detection system of the present invention.

[0041] (1) The plasma concentration of PTN in the above samples was detected by ELISA. The specific steps are as follows: ① Sample incubation: Add 100 μL of standards of different concentrations and pretreated test samples to each well, cover with sealing tape, and incubate at 37℃ in the dark for 1.5 h. After incubation, add 300 μL of 1× washing buffer to each well, gently shake for 30 s, shake dry and pat dry on paper. Repeat this washing process 3 times.

[0042] ② Antibody incubation: Add 100 μL of biotinylated antibody working solution to each well, mix gently, cover with sealing tape, and incubate at 37°C in the dark for 1 h. After incubation, repeat the washing process in step 2 four times.

[0043] ③ Enzyme label incubation: Add 100 μL of 1×SA-HRP working solution to each well, cover with sealing tape, incubate at 37℃ in the dark for 30 min, wash 4 times, and pat dry.

[0044] ④ Substrate color development: First, add 50 μL of color development solution A to each well, followed by 50 μL of color development solution B. Mix gently, cover with sealing tape, and incubate at 37°C in the dark for 15 min. (Adjust the color development time according to the color of the sample and control antibody.) ⑤ Termination of reaction: After the colorimetric reaction is complete, add 50 μL of stop solution to each well, mix gently, and measure the absorbance at 450 nm using a preheated microplate reader within 5 minutes.

[0045] (2) Establish a PTN standard curve and perform concentration conversion; (3) Perform statistical analysis on the test results. For the four proteins IL12, CCL3, CASP-8, and ADGRG1, based on the sequencing results and ROC analysis of the Olink platform in Example 1, the same ELISA detection procedure as PTN can be used for detection, and they can be used together with PTN to construct a joint diagnostic model.

[0046] III. Experimental Results Preoperative plasma concentrations of PTN in breast cancer patients were significantly higher than those in healthy controls, while postoperative plasma concentrations decreased significantly compared to preoperative levels (statistical difference). Figure 5 PTN (particulate transluminal tract marker) showed good distinguishing ability between preoperative samples and healthy controls, with a high AUC (abnormal oscillatory value). (See attached image.) Figure 6 The above results demonstrate that PTN possesses real and stable clinical testing feasibility.

[0047] Example 4: Immunohistochemical histological verification I. Samples and Materials Paraffin-embedded tissue sections from surgically removed specimens of breast cancer patients were collected. The paraffin sections were 4 μm thick and consisted of 3 pairs of paired samples, including: breast cancer tissue sections (tumor tissue) and adjacent normal tissue sections (morphologically normal breast tissue 5 cm from the tumor margin).

[0048] II. Experimental Methods (1) Dewaxing and hydration First, the paraffin sections were baked in a 60°C oven for about 45 minutes. Then, they were dewaxed by treating with xylene I and II for 10 minutes each. Next, they were subjected to gradient ethanol hydration: 100 vt% ethanol for 5 minutes twice; 95 vt% ethanol for 5 minutes; 80 vt% ethanol for 5 minutes; 70 vt% ethanol for 5 minutes. Finally, they were rinsed with distilled water and washed with PBS for later use.

[0049] (2) Antigen retrieval The hydrated slices were placed in a citrate solution at pH 6.0, microwaved to boiling and held for 12 minutes, then allowed to cool naturally. The slices were then washed three times with PBS for 4 minutes each time.

[0050] (3) Blocking endogenous peroxidase Add 3 vt% hydrogen peroxide solution, incubate at room temperature for 10 min, and wash 3 times with PBS.

[0051] (4) Blocking nonspecific binding Add protein blocking solution and incubate at room temperature for 25 minutes. Discard the blocking solution without washing.

[0052] (5) Primary antibody incubation Add PTN primary antibody according to the instructions, incubate overnight at 4°C, and wash three times with PBS the next day.

[0053] (6) Secondary antibody incubation Add HRP-labeled secondary antibody, incubate at room temperature for 25 min, and wash three times with PBS.

[0054] (7) DAB color development and restaining First, add DAB developing solution and observe the color development. Once the desired intensity is reached, stop the staining with tap water. Then, add hematoxylin for counterstaining for 45 seconds and rinse with running water.

[0055] (8) Dehydration, clearing and sealing The slides were dehydrated using a gradient of ethanol at concentrations of 70 vt%, 80 vt%, 95 vt%, and 100 vt%, followed by clearing with xylene twice. Finally, the slides were mounted with neutral resin, dried, and examined under a microscope and photographed.

[0056] III. Experimental Results Experimental results are as follows Figure 7 As shown, PTN was significantly highly expressed in breast cancer tissue, with statistically significant differences, consistent with the trend of plasma detection results.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of multi-protein biomarkers in the preparation of breast cancer therapeutic drugs or early screening kits, characterized in that, The multi-protein biomarkers include pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, caspase 8, and adhesion G protein-coupled receptor G1.

2. The application according to claim 1, characterized in that, Among the multi-protein biomarkers, pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, and adhesion G protein-coupled receptor G1 were upregulated in the plasma of breast cancer patients, while caspase-8 was downregulated in the plasma of breast cancer patients.

3. A breast cancer early screening kit, characterized in that, It includes a capture antibody component, a detection antibody component, a standard component, auxiliary reagents, and an interpretation module; The capture antibody component includes specific capture antibodies against pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, caspase 8, and adhesion G protein-coupled receptor G1, respectively. The detection antibody component includes corresponding detection antibodies for pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, caspase 8, and adhesion G protein-coupled receptor G1. The standard component includes pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, caspase 8, and adhesion G protein-coupled receptor G1 standard protein at known concentration gradients.

4. The breast cancer early screening kit according to claim 3, characterized in that, The auxiliary reagents include blocking solution, washing solution, chromogenic substrate, stop solution, and reaction buffer.

5. The breast cancer early screening kit according to claim 3, characterized in that, The interpretation module includes an instruction manual or software that converts the test results into a diagnostic score and provides a reference threshold.

6. A method for establishing an early breast cancer screening system, characterized in that, Includes the following steps: S1. Collect peripheral blood samples from breast cancer patients and normal individuals, and separate plasma or serum as the test samples; S2. The expression levels of pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, caspase 8 and adhesion G protein-coupled receptor G1 in the test sample obtained in S1 are detected using the kit described in any one of claims 3-5, and the detection results are obtained. S3. Construct a joint diagnostic model based on the detection results obtained in S3, and determine the diagnostic cutoff value.

7. The method for establishing an early breast cancer screening system according to claim 6, characterized in that, The joint diagnostic model in S3 is a linear or nonlinear regression model, including the following forms: Score=α1×PTN+α2×IL12+α3×CCL3+α4×CASP-8+α5×ADGRG1+β; Among them, α1-α5 are the weight coefficients of pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, caspase 8 and adhesion G protein-coupled receptor G1, respectively. PTN, IL12, CCL3, CASP-8, and ADGRG1 are the detection results of pleiotropic neurotrophic factor, interleukin-12, macrophage inflammatory protein 1-α, caspase 8, and adhesion G protein-coupled receptor G1, respectively. β is a constant term; Score is the diagnostic score; When the diagnostic score is greater than or equal to the diagnostic cutoff value, the subject is determined to be a high-risk individual for breast cancer; When the diagnostic score is less than the diagnostic cutoff value, the subject is considered a low-risk individual for breast cancer.

8. The method for establishing an early breast cancer screening system according to claim 6 or 7, characterized in that, The diagnostic cutoff values ​​are determined based on training samples using statistical or machine learning methods, including: 1) The point where the Youden exponent is at its maximum on the ROC curve; 2) The dividing point under preset sensitivity or specificity conditions; 3) The optimal threshold obtained through cross-validation or independent validation arrays.

9. A breast cancer early screening system, characterized in that, Obtained by the method described in any one of claims 6-8.

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