Kit for predicting colorectal cancer liver metastasis risk and prognosis

Abstract

The invention relates to a kit for predicting colorectal cancer liver metastasis risk and prognosis, and belongs to the technical field of biomedicine. The kit comprises: (a) an antibody for specifically recognizing phosphoglycerate kinase 1 (PGK1) protein; and (b) a scoring reference for semi-quantitative scoring of PGK1 protein immunohistochemical staining results. The invention provides a colorectal cancer liver metastasis risk and prognosis evaluation solution which is accurate in prediction, easy to popularize, new in mechanism and wide in application, effectively overcomes the defects of insufficient sensitivity, low specificity and lack of molecular mechanism basis of the existing clinical prediction means, has outstanding substantive features and remarkable progress, and has a wide application prospect. The social benefit and the economic benefit are obvious.

Description

Technical Field

[0001] This invention relates to a kit for predicting the risk and prognosis of colorectal cancer liver metastasis, belonging to the field of biomedical technology. Background Technology

[0002] Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Epidemiological data shows that CRC is the second leading cause of cancer-related deaths, with liver metastasis (CRLM) being the most common and deadliest form of distant metastasis. Approximately 50% of CRC patients develop liver metastases during the course of their disease, and the five-year survival rate for these patients is significantly lower than that for those without metastases. Therefore, early identification of high-risk individuals and accurate prediction of liver metastasis are significant challenges in CRC management.

[0003] Currently, commonly used biomarkers in clinical practice, such as CEA and CA19-9, while playing a role in the screening and follow-up of CRC, have limited sensitivity and specificity in the early warning of CRLM, making it difficult to meet the clinical needs for precise prediction and personalized intervention. With the development of multi-omics technologies, more and more studies are focusing on the role of metabolic reprogramming in the tumor metastasis process, especially the mechanism by which tumor cells adapt to the heterogeneous metastatic microenvironment by enhancing glycolytic activity. However, the specific functions of key glycolytic enzymes in CRLM and their potential as prospective predictive indicators still require further investigation.

[0004] Phosphoglycerate kinase 1 (PGK1) is one of the key rate-limiting enzymes in the glycolysis pathway, catalyzing the conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate, accompanied by ATP production. In recent years, PGK1 has been found to play a role not only in energy metabolism but also in regulating processes such as tumor cell migration and extracellular matrix remodeling. In tumor types such as breast cancer and kidney cancer, high expression of PGK1 is closely associated with metastasis and poor prognosis. However, whether its expression level in CRC, especially in the primary tumor, can predict the risk of liver metastasis remains insufficiently understood.

[0005] Currently, no systematic research has been found in publicly available literature or patent databases on the association between PGK1 expression levels in primary CRC lesions and the risk of future liver metastasis, nor on any reports of technical studies translating this association into predictive schemes with clearly defined cutoff values. Existing research has the following limitations: Firstly, the limited research involving PGK1 and CRC metastasis primarily focuses on retrospective descriptions of established metastatic lesions. However, obtaining paired, high-quality clinical specimens of CRC liver metastases is extremely difficult (limited by surgical indications, specimen availability, etc.), resulting in small sample sizes in such studies (e.g., only a few cases), making it difficult to draw statistically valid conclusions, let alone serve as a predictor of pre-metastasis occurrence. Secondly, existing technologies largely concentrate on exploring the function of PGK1 in other solid tumors, lacking specific molecular mechanism-based predictive indicators for the specific scenario of CRC liver metastasis.

[0006] Furthermore, existing predictive tools largely rely on clinicopathological information or traditional tumor markers, lacking specific predictive indicators based on molecular mechanisms. These tools cannot effectively identify high-risk CRC patients who have not yet developed liver metastases, nor can they dynamically monitor trends in metastasis risk, limiting the possibility of individualized management and early intervention. Therefore, there is an urgent need to develop a CRLM predictive kit based on reliable molecular indicators, possessing both high sensitivity and specificity, capable of providing high early warning value at the primary stage, thus offering a more guiding risk assessment tool for clinical practice.

[0007] While there are currently immunohistochemical (IHC) detection kits for PGK1 protein available on the market for research purposes, their use is limited to general qualitative or semi-quantitative detection of PGK1 protein. They are not associated with any specific disease prognosis model or risk threshold, nor do they cover specific application methods and diagnostic cutoff values ​​in predicting the risk of liver metastasis from primary CRC lesions. Summary of the Invention

[0008] The first objective of this invention is to provide a kit for predicting the risk and prognosis of colorectal cancer liver metastasis.

[0009] To achieve the first objective of the present invention, the kit comprises:

[0010] (a) An antibody that specifically recognizes the phosphoglycerate kinase 1 (PGK1) protein; and,

[0011] (b) Scoring reference used for semi-quantitative scoring of PGK1 protein immunohistochemical staining results.

[0012] The PGK1 is phosphoglycerate kinase 1.

[0013] In one specific embodiment, the kit further includes a secondary antibody-enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer.

[0014] In one specific embodiment, the kit further includes: hematoxylin, DAB chromogenic solution, and pH 6.0 sodium citrate buffer.

[0015] In one specific embodiment, the kit further includes: 4% paraformaldehyde, 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, xylene, paraffin, 3% H2O2, and normal animal serum diluted 20:1.

[0016] In one specific implementation, the scoring reference includes standard stained images corresponding to different scoring levels, and / or records preset thresholds for risk assessment based on the scoring results.

[0017] In one specific implementation, the preset threshold is a PGK1 expression semi-quantitative score of 0.579;

[0018] The scoring method is as follows: Total score = A × B, where A is the percentage of positive cells, and the value of A is 0-1; B is the staining intensity level, with a value of 0 for negative staining intensity, 1 for weak positive staining intensity, 2 for medium positive staining intensity, and 3 for strong positive staining intensity.

[0019] The standard staining images corresponding to the different scoring levels include: negative standard staining images, weakly positive standard staining images, moderately positive standard staining images, and strongly positive standard staining images.

[0020] In one specific embodiment, the antibody is an anti-PGK1 monoclonal antibody.

[0021] A second objective of this invention is to provide the use of the above-described kit in the preparation of a tool for assisting in the assessment of the risk and prognosis of liver metastases in patients with colorectal cancer.

[0022] To achieve the second objective of this invention, the above-described kit is used to prepare a tool for assisting in the assessment of liver metastasis risk and prognosis in colorectal cancer patients.

[0023] In one specific embodiment, the method for assisting in the assessment of liver metastasis risk and prognosis in colorectal cancer patients includes the following steps:

[0024] a. Provide tumor tissue samples from colorectal cancer patients;

[0025] b. Use the above-described kit to detect the expression level of PGK1 protein in the tumor tissue sample and obtain a semi-quantitative score; c. Compare the score with a preset risk threshold to obtain the PGK1 expression risk level information of the sample.

[0026] In one specific implementation, the preset risk threshold is 0.579.

[0027] Beneficial effects:

[0028] Compared with the prior art, the technical solution provided by the present invention has the following significant advantages:

[0029] 1. Significantly Improved Predictive Performance, Achieving Early Risk Warning Based on Primary Lesion: The core innovation of this invention lies in the first-ever quantitative correlation between the expression level of PGK1 protein and the risk of future liver metastasis in patients based on primary colorectal cancer tissue. A diagnostic threshold with high predictive efficacy (e.g., IHC score ≥ 0.579) has been established and validated with a large sample size. ROC curve analysis shows that its predictive efficacy (AUC = 0.715) is significantly superior to that of traditional serum markers such as CEA and CA19-9 in early metastasis prediction. This allows clinicians to identify high-risk patients through routine pathological analysis in the early stages, when only primary lesion specimens are available and imaging has not yet revealed metastases. This achieves truly prospective early warning, gaining valuable time for early intervention. The main conclusions and predictive value of this invention are derived from rigorous statistical analysis of the primary lesion cohort. The limited metastasis data mentioned in this paper are only supplementary evidence for mechanistic exploration.

[0030] 2. The detection method has strong clinical applicability and is easy to promote and popularize: This invention is based on the immunohistochemistry (IHC) technology platform that is routinely used in clinical pathology departments, without the need to introduce expensive and complex special equipment (such as gene sequencers). The provided reagent kits and supporting scoring standards have standardized operating procedures, low cost, and low technical threshold, making them very easy to standardize and apply in hospitals at all levels, especially primary healthcare institutions, and have good prospects for clinical translation and market feasibility.

[0031] 3. Novel biomarker mechanism, directly linked to metastatic potential: This invention establishes PGK1 for the first time as a predictive biomarker for colorectal cancer liver metastasis based on the primary tumor site. Unlike traditional serum biomarkers such as CEA, which rely on indirect monitoring of tumor-released substances, PGK1, as a glycolytic metabolic enzyme, directly drives metabolic reprogramming through high expression in primary tumor cells, enhancing the cells' invasive and metastatic adaptation capabilities. Therefore, detecting PGK1 levels in the primary tumor site assesses metastatic risk from the perspective of the intrinsic driving mechanism of tumor cells, providing a novel and more direct biological predictive dimension.

[0032] 4. Providing key decision-making basis for personalized precision medicine: The predictive results of this invention can be directly used for clinical decision support. For patients with high-risk scores, clinicians can consider increasing the frequency of postoperative follow-up, conducting more precise imaging assessments earlier (such as enhanced MRI), or recommending preventive / adjuvant therapy (such as hepatic artery infusion chemotherapy) after comprehensive evaluation. Conversely, for low-risk patients, excessive examinations and treatments can be avoided, reducing the physical and mental burden and economic pressure on patients, and truly achieving differentiated and individualized treatment strategies.

[0033] 5. Prognostic Value and Broad Application Prospects: In addition to predicting metastasis risk, this invention, through survival analysis using public databases (such as TCGA), confirms that high PGK1 expression in the primary lesion is significantly associated with shortened overall survival (OS). Therefore, this detection method can not only be used for preoperative / postoperative metastasis risk stratification, but also serve as an independent prognostic indicator for assessing the overall disease progression and survival outcome of patients, possessing significant application value in clinical research, efficacy evaluation, and comprehensive patient management.

[0034] In summary, this invention provides a novel and accurate solution for assessing the risk and prognosis of colorectal cancer liver metastasis based on primary lesion detection. It effectively overcomes the shortcomings of existing methods, such as insufficient sensitivity in early warning and lack of prospective molecular mechanism evidence. Its core innovation and practicality lie in the discovery and application of predictive value for primary lesions, demonstrating significant substantive features and remarkable progress, resulting in substantial social and economic benefits. Attached Figure Description

[0035] Figure 1 Immunohistochemical staining images of PGK1 in three groups of tissue samples in Example 1.

[0036] Figure 2 Statistical chart of immunohistochemical scores in Example 1.

[0037] Figure 3 : Receiver operating characteristic curve of Example 1.

[0038] Figure 4 Example 2: PGK1 expression levels in colonic adenocarcinoma (COAD) and rectal adenocarcinoma (READ) from the TCGA database;

[0039] Figure 5 Example 3: Relationship between PGK1 expression in the GEO database and patient prognosis. Detailed Implementation

[0040] To achieve the first objective of the present invention, the kit comprises:

[0041] (a) An antibody that specifically recognizes the phosphoglycerate kinase 1 (PGK1) protein; and,

[0042] (b) Scoring reference used for semi-quantitative scoring of PGK1 protein immunohistochemical staining results.

[0043] The PGK1 is phosphoglycerate kinase 1.

[0044] In one specific embodiment, the kit further includes a secondary antibody-enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer.

[0045] In one specific embodiment, the kit further includes: hematoxylin, DAB chromogenic solution, and pH 6.0 sodium citrate buffer.

[0046] In one specific embodiment, the kit further includes: 4% paraformaldehyde, 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, xylene, paraffin, 3% H2O2, and normal animal serum diluted 20:1.

[0047] In one specific implementation, the scoring reference includes standard stained images corresponding to different scoring levels, and / or records preset thresholds for risk assessment based on the scoring results.

[0048] In one specific implementation, the preset threshold is a PGK1 expression semi-quantitative score of 0.579;

[0049] The scoring method is as follows: Total score = A × B, where A is the percentage of positive cells, and the value of A is 0-1; B is the staining intensity level, with a value of 0 for negative staining intensity, 1 for weak positive staining intensity, 2 for medium positive staining intensity, and 3 for strong positive staining intensity.

[0050] The standard staining images corresponding to the different scoring levels include: negative standard staining images, weakly positive standard staining images, moderately positive standard staining images, and strongly positive standard staining images.

[0051] In one specific embodiment, the antibody is an anti-PGK1 monoclonal antibody.

[0052] In one specific embodiment, the method includes the following steps:

[0053] ① Sample collection and preparation: Tissue specimens were collected from pathologically confirmed CRC patients. After formalin fixation and paraffin embedding, the specimens were prepared into serial sections for IHC detection.

[0054] ②PGK1 expression detection: Commercially available anti-PGK1 antibody was used for immunostaining according to standard IHC staining procedures. The PGK1 staining intensity and the proportion of positive cells were comprehensively evaluated using a semi-quantitative scoring system to determine the final PGK1 expression level.

[0055] ③ Data analysis: Statistical analysis was performed on the expression level of PGK1 and the liver metastasis status of patients.

[0056] To achieve the second objective of this invention, the above-described kit is used to prepare a tool for assisting in the assessment of liver metastasis risk and prognosis in colorectal cancer patients.

[0057] In one specific embodiment, the method for preparing a treatment to assist in assessing the risk and prognosis of liver metastasis in colorectal cancer patients includes the following steps:

[0058] a. Provide tumor tissue samples from colorectal cancer patients;

[0059] b. Use the above-described kit to detect the expression level of PGK1 protein in the tumor tissue sample and obtain a semi-quantitative score; c. Compare the score with a preset risk threshold to obtain the PGK1 expression risk level information of the sample.

[0060] In one specific implementation, the preset risk threshold is 0.579.

[0061] The specific embodiments of the present invention will be further described below with reference to examples, but the present invention is not limited to the scope of the embodiments described herein.

[0062] Example 1

[0063] 1. Experimental Design and Sample Composition

[0064] This study included tissue specimens from 90 clinical CRC patients, divided into three groups: no liver metastasis group (n=38): primary lesion tissue, patients without liver metastasis; liver metastasis group (n=38): primary lesion tissue, patients with liver metastasis at the time of diagnosis; liver metastasis group (n=14) (liver metastasis samples were difficult to obtain, and metastasis data were only for auxiliary verification): liver metastasis specimens obtained from patients during the same period.

[0065] 2. Immunohistochemical (IHC) staining method

[0066] PGK1 expression levels were obtained by immunohistochemical detection, and the experimental procedures followed the standard protocol:

[0067] ① Tissue fixation and dehydration embedding: Freshly excised CRC tissue was fixed in 4% paraformaldehyde for 48 hours, rinsed with running water for 1–2 hours, dehydrated in 75%, 85%, 95%, and 100% ethanol in sequence, and then embedded in paraffin after treatment with xylene.

[0068] ② Sectioning and drying: Use a microtome to section the paraffin tissue to a thickness of 5 μm, set the spreading temperature to 65℃, and dry it on a glass slide. Store at room temperature for later use.

[0069] ③ Dewaxing and hydration: After baking the slices at 65℃, they are sequentially dewaxed and hydrated in xylene and 100%~75% ethanol, and finally rinsed in distilled water.

[0070] ④ Blocking endogenous peroxidase: Place the slices in 3% H2O2 and incubate in the dark for 10 minutes to block the endogenous enzyme activity.

[0071] ⑤ Antigen retrieval: The high-pressure heat retrieval method was used, which involved boiling the antigen in a pH 6.0 sodium citrate buffer solution under high pressure and then allowing it to cool to room temperature.

[0072] ⑥ Blocking and antibody incubation: Block non-specific sites with normal animal serum diluted 20:1 and incubate at 37°C for 20 minutes, then add PGK1 primary antibody and incubate overnight at 4°C.

[0073] ⑦ Secondary antibody incubation and color development: After incubating with secondary antibody at room temperature for 1 hour, use DAB for color development. Control the termination time according to the color appearance. Rinse with tap water to terminate the reaction.

[0074] ⑧ Nuclear staining and mounting: After hematoxylin counterstaining and blueing treatment, the slides are dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin, and finally scanned for imaging.

[0075] ⑨ IHC Scoring Criteria: PGK1 staining results are semi-quantitatively scored using the following formula: Total Score = A × B. Where A is the percentage of positive cells (0-1), and B is the staining intensity grade (0=negative, 1=weakly positive, 2=moderately positive, 3=strongly positive). The final score range is 0-3.

[0076] 3. Experimental Results

[0077] This invention evaluated the expression differences of PGK1 in different CRC metastatic states through IHC staining and quantitative analysis to verify its effectiveness as a predictive biomarker for liver metastasis. Figure 1The immunohistochemical staining of PGK1 in three groups of tissue samples is shown. Overall morphological observation reveals the following: Primary lesion without liver metastases (left): PGK1 staining signal is weak, with localized light brown staining, indicating overall low expression; Primary lesion with liver metastases (middle): PGK1 staining is moderate, with widespread moderate brown deposits in the cytoplasm; Liver metastases (right): PGK1 staining is most prominent, with extensive dark brown staining in the tumor tissue, suggesting high expression in metastatic lesions.

[0078] Figure 2 The corresponding immunohistochemical scoring statistics are shown in the figure. The data were calculated based on the PGK1 staining intensity and the proportion of positive cells in each group. One-way ANOVA was used for inter-group comparisons, and the results are as follows: Statistically significant differences were found between groups: liver metastasis group vs. primary lesion with liver metastasis group: p < 0.0001; liver metastasis group vs. primary lesion without metastasis group: p < 0.0001; primary lesion with liver metastasis group vs. primary lesion without metastasis group: p = 0.0062. These results indicate that PGK1 expression level is closely related to the liver metastasis status of CRC. Particularly noteworthy is the significant increase in PGK1 expression in primary lesions with liver metastasis, suggesting its potential to reflect metastasis risk early.

[0079] To evaluate the diagnostic efficacy of PGK1 expression levels in predicting CRLM, we performed receiver operating characteristic (ROC) curve analysis on samples from two clinical groups—"primary lesions without liver metastases" and "primary lesions with liver metastases"—based on IHC staining scores. ROC curves are an important tool for evaluating the performance of classification models, revealing the discriminative ability of biomarkers by balancing sensitivity and specificity at different cutoff values.

[0080] Figure 3 ROC analysis showed an area under the curve (AUC) of 0.715, indicating that PGK1 expression has moderate to high discriminative ability in distinguishing between the two groups of tumor tissues. The best cutoff value was 0.579, meaning that when PGK1 staining score threshold is set at 0.579, the sum of sensitivity and specificity is maximized, which can serve as a quantitative basis for predicting the risk of colorectal cancer liver metastasis. These results confirm that the tissue expression level of PGK1 has good predictive value and has clinical application potential in determining whether colorectal cancer is accompanied by liver metastasis. Especially for patients with early liver metastases that are difficult to detect through imaging, the PGK1 IHC score, as a tissue-derived, stable, and reliable predictive tool, can provide important information for auxiliary judgment.

[0081] Example 2

[0082] Analysis of transcriptomic data from colorectal cancer patients in the TCGA (The Cancer Genome Atlas) database revealed that PGK1 expression levels in colorectal cancer tissues were significantly higher than in adjacent normal tissues. Specifically, in both the colonic adenocarcinoma (COAD) and rectal adenocarcinoma (READ) subtypes, PGK1 expression levels were higher than in their corresponding normal tissues (e.g., normal normal tissues). Figure 4 As shown in the figure, red represents cancerous tissue and gray represents adjacent tissue. The difference was statistically significant (p < 0.05). This result suggests that elevated PGK1 expression may be closely related to the occurrence and progression of colorectal cancer.

[0083] Example 3

[0084] Analysis of the relationship between PGK1 expression and patient prognosis using survival data from the GEO database showed that patients with high PGK1 expression had significantly shorter overall survival (OS) than those with low expression (see...). Figure 5 The hazard ratio (HRR) for the high expression group was 1.3 (95% CI: 1.02–1.65), with a Log-rank test p=0.032. This indicates that high PGK1 expression is not only associated with tumorigenesis at the tissue level, but also significantly correlated with poor prognosis at the clinical level.

[0085] in conclusion:

[0086] This invention, through clinical sample testing and validation using a large-scale public database, comprehensively demonstrates the scientific validity, practicality, and clinical application value of PGK1 as a risk prediction biomarker for colorectal cancer metastasis (CRLM). In tissue-level immunohistochemical (IHC) experiments, this invention constructed a standardized PGK1 detection procedure, applied to 90 tissue specimens from primary lesions without liver metastases, primary lesions with liver metastases, and liver metastases. Results showed that PGK1 expression levels increased with increasing liver metastasis risk, with stable and clear staining signals. After quantitative scoring, ROC curve analysis indicated that PGK1 had good discriminative performance in distinguishing between colorectal cancer patients with and without liver metastases (AUC = 0.715), with an optimal cutoff value of 0.579, suggesting that this indicator possesses a clear risk identification capability.

[0087] Further analysis of a large sample of colorectal cancer patients using the TCGA database revealed that PGK1 expression in cancerous tissues was significantly higher than in adjacent normal tissues (P < 0.05), and its high expression was significantly associated with shorter overall survival, demonstrating good prognostic ability. This conclusion not only supports the possibility that PGK1 participates in the metastatic process at the mechanistic level, but also verifies its universality as a risk marker at the population level.

[0088] In summary, this invention fully validates the effectiveness and applicability of PGK1 in predicting CRLM. This method can be implemented using a conventional IHC platform, offering advantages such as ease of operation and low cost. It also possesses high sensitivity and specificity, playing a crucial role in postoperative specimen analysis, follow-up management, and adjuvant treatment decision-making, providing important technical support for promoting the early identification and precise intervention of CRLM.

Claims

1. A kit for predicting the risk and prognosis of colorectal cancer liver metastasis, characterized in that, The kit contains: (a) An antibody that specifically recognizes the PGK1 protein; and, (b) Scoring reference used for semi-quantitative scoring of PGK1 protein immunohistochemical staining results.

2. The kit for predicting the risk and prognosis of colorectal cancer liver metastasis according to claim 1, characterized in that, The kit also includes a secondary antibody-enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer.

3. The kit for predicting the risk and prognosis of colorectal cancer liver metastasis according to claim 1, characterized in that, The kit also includes: hematoxylin, DAB chromogenic solution, and pH 6.0 sodium citrate buffer.

4. The kit for predicting the risk and prognosis of colorectal cancer liver metastasis according to any one of claims 1 to 3, characterized in that, The kit also includes: 4% paraformaldehyde, 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, xylene, paraffin, 3% H2O2, and normal animal serum diluted 20:

1.

5. The kit for predicting the risk and prognosis of colorectal cancer liver metastasis according to claim 1, characterized in that, The scoring references include standard stained images corresponding to different scoring levels, and / or record preset thresholds for risk assessment based on the scoring results.

6. The kit for predicting the risk and prognosis of colorectal cancer liver metastasis according to claim 5, characterized in that, The preset threshold is 0.579, which is the semi-quantitative score of PGK1 expression. The scoring method is as follows: Total score = A × B, where A is the percentage of positive cells, and the value of A is 0-1; B is the staining intensity level, with a value of 0 for negative staining intensity, 1 for weak positive staining intensity, 2 for medium positive staining intensity, and 3 for strong positive staining intensity. The standard staining images corresponding to the different scoring levels include: negative standard staining images, weakly positive standard staining images, moderately positive standard staining images, and strongly positive standard staining images.

7. The kit for predicting the risk and prognosis of colorectal cancer liver metastasis according to any one of claims 1 to 6, characterized in that, The antibody is an anti-PGK1 monoclonal antibody.

8. Use of the kit according to any one of claims 1 to 7 in the preparation of a tool for assisting in the assessment of the risk and prognosis of liver metastasis in patients with colorectal cancer.

9. The use according to claim 8, characterized in that, Methods used to assist in assessing the risk of liver metastasis and prognosis in colorectal cancer patients include the following steps: a. Provide tumor tissue samples from colorectal cancer patients; b. Using the kit according to any one of claims 1 to 7, detect the expression level of PGK1 protein in the tumor tissue sample and obtain a semi-quantitative score; c. Compare the score with a preset risk threshold to obtain the PGK1 expression risk level information of the sample.

10. The use according to claim 9, characterized in that, The preset risk threshold is 0.579.

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