Marker for predicting the risk of distant metastasis after surgery in colorectal cancer patients and use thereof

Abstract

The application provides a marker for predicting the risk of distant metastasis of a colorectal cancer patient after surgery and an application thereof, and belongs to the technical field of molecular biology and medicine.The marker for predicting the risk of distant metastasis of a colorectal cancer patient after surgery according to the application is composed of FHL3 protein, GGA1 protein, NUDFS7 protein and TGFBI protein.The expression levels of the four protein molecules can reflect the biological characteristics of the colorectal cancer patient, so that the risk of distant metastasis of the colorectal cancer patient after surgery can be better predicted, and the treatment of the colorectal cancer patient in the II or III stage can be better guided.

Description

Technical Field

[0001] This invention belongs to the field of molecular biomedical technology, specifically relating to biomarkers for predicting the risk of distant metastasis after surgery in colorectal cancer patients and their applications. Background Technology

[0002] Malignant tumors, particularly colorectal cancer, pose a serious threat to the health of the Chinese people. The latest data from the World Health Organization shows that 20% of new cancer cases and 24% of cancer deaths worldwide occur in China. Approximately 3.12 million new cancer cases are diagnosed in my country annually, while annual healthcare expenditure on cancer treatment reaches US$10 billion. Colorectal cancer ranks third in incidence and fifth in mortality among malignant tumors in my country. Adjuvant radiotherapy and chemotherapy are crucial treatment methods for colorectal cancer patients. Our initial focus is on colorectal cancer, a prevalent cancer in my country. By constructing a multi-level characteristic spectrum of colorectal cancer and establishing a model for evaluating the efficacy of chemotherapy in colorectal cancer, we can guide individualized clinical treatment plans for colorectal cancer, improve the overall treatment level of colorectal cancer, and bring certain socio-economic benefits.

[0003] Clinically, stage II and III colorectal cancer patients are usually treated surgically, but some still develop distant metastases after surgery, leading to poor prognoses. Currently, there is a lack of specific molecular markers for predicting postoperative distant metastasis in colorectal cancer. Therefore, researching molecules that can predict postoperative metastasis in colorectal cancer is a significant task.

[0004] Proteins are the bearers and manifestations of life activities. With the discovery of mass spectrometry, tumor proteomics analysis has become a promising method for identifying prognostic and predictive biomarkers for invasiveness, survival outcomes, and treatment monitoring. While previous proteomics studies have been used to generate differentially expressed biomarkers between colorectal cancer and non-cancerous tissues, the differences in proteomics analysis between patients with different prognoses have largely remained unexplored. Furthermore, due to small sample sizes, a lack of large-scale validation cohorts, and standardized procedures, few proteomic biomarkers have been applied in clinical practice. Summary of the Invention

[0005] In response to the above problems, the purpose of this invention is to provide biomarkers for predicting the risk of distant metastasis after surgery in patients with colorectal cancer and their applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a set of biomarkers for predicting the risk of distant metastasis after surgery in colorectal cancer patients, consisting of FHL3 protein, GGA1 protein, NUDFS7 protein and TGFBI protein.

[0007] In a preferred embodiment of the biomarker for predicting the risk of distant metastasis after surgery in patients with colorectal cancer according to the present invention, the colorectal cancer is stage II or stage III colorectal cancer.

[0008] The present invention also provides the use of the biomarkers for predicting the risk of postoperative distant metastasis in colorectal cancer patients in the preparation of reagents for predicting the risk of postoperative distant metastasis in colorectal cancer patients.

[0009] As an application of the present invention, the reagents include those for detecting the expression levels of FHL3 protein, GGA1 protein, NUDFS7 protein and TGFBI protein in tumor tissue using mass spectrometry-based proteomics technology, multiplex immunohistochemistry technology combined with artificial intelligence automated quantification technology.

[0010] This invention also provides a method for constructing a prediction model for the risk of distant metastasis after surgery in patients with colorectal cancer, comprising the following steps: (1) Obtaining differentially expressed proteins: Collect the test samples, extract the whole protein from the tissue, and use mass spectrometry analysis, Wilcox test analysis, LASSO logistic algorithm and SVM logistic model to analyze and screen the extracted whole protein to obtain differentially expressed proteins; (2) Construct a prediction model for the risk of distant metastasis after surgery in colorectal cancer patients based on differentially expressed proteins: The expression level of the differentially expressed proteins obtained in step (1) is detected, the proportion of tumor cells expressing the differentially expressed proteins in the tissue is obtained, and the proportion of each cell is substituted into the calculation formula to obtain the risk score; the risk of distant metastasis after surgery in colorectal cancer patients is judged based on the risk score.

[0011] In a preferred embodiment of the method for constructing the postoperative distant metastasis risk prediction model for colorectal cancer patients according to the present invention, the differentially expressed proteins include FHL3 protein, GGA1 protein, NUDFS7 protein and TGFBI protein.

[0012] In a preferred embodiment of the method for constructing the postoperative distant metastasis risk prediction model for colorectal cancer patients according to the present invention, the tumor cells include FHL3+ tumor cells, GGA1+NUDFS7+ tumor cells and TGFBI+ tumor stromal cells.

[0013] As a preferred embodiment of the method for constructing the postoperative distant metastasis risk prediction model for colorectal cancer patients described in this invention, the calculation formula is: Risk score = 1.500 × FHL3 + tumor cell composition ratio (%) - 2.552 × GGA1 + NDUFS7 + tumor cell composition ratio (%) + 1.704 × TGFBI + tumor stromal cell composition ratio (%).

[0014] In a preferred embodiment of the method for constructing the postoperative distant metastasis risk prediction model for colorectal cancer patients according to the present invention, the threshold value of the risk score is 0.978. If the risk score value is ≤0.978, the sample is judged to be at low risk of postoperative distant metastasis; if it is >0.978, the sample is judged to be at high risk of postoperative distant metastasis.

[0015] The present invention also provides a prediction model for the risk of distant metastasis after surgery in colorectal cancer patients, constructed according to the method for constructing the prediction model for the risk of distant metastasis after surgery in colorectal cancer patients.

[0016] The beneficial effects of this invention are as follows: This invention provides biomarkers and their applications for predicting the risk of distant metastasis after surgery in colorectal cancer patients. This invention obtains a set of biomarkers that can be used to predict the risk of distant metastasis after surgery in colorectal cancer patients through analysis and screening. It uses mass spectrometry-based proteomics technology, multiplex immunohistochemistry technology combined with artificial intelligence automated quantification technology to detect the expression levels of protein molecules FHL3, GGA1, NUDFS7, and TGFBI in their tumor tissues. These biomarkers are then substituted into a specific calculation formula to calculate the risk score of distant metastasis after surgery in stage II or III colorectal cancer patients, thereby predicting the risk of distant metastasis after surgery in colorectal cancer patients and better guiding the treatment of stage II or III colorectal cancer patients. Attached Figure Description

[0017] Figure 1 Kaplan-Meier analysis of disease-free survival rates in colorectal cancer patients in the training group, internal validation group, and external validation group; Figure 2 ROC curves for the specificity and sensitivity analysis of each model in predicting distant metastasis after surgery in patients with stage II and III colorectal cancer. Detailed Implementation

[0018] To more concisely and clearly demonstrate the technical solution, purpose, and advantages of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0019] Example 1 This embodiment analyzes and screens biomarkers for predicting the risk of distant metastasis after surgery in patients with colorectal cancer. The specific method is as follows: Sixty patients with stage II or III colorectal cancer were selected, including 30 patients who developed distant metastasis after surgery and the remaining 30 patients who did not develop distant metastasis after surgery. Tumor tissue and paired adjacent normal colorectal tissue (normal colorectal mucosa) were collected from these patients. After whole-protein extraction, denaturation and reduction, protein alkylation, enzymatic digestion, peptide desalting, and separation, the samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). For the raw mass spectrometry data, using software programs such as Mascot, and with a false discovery rate (FDR) controlled to be less than 1%, a total of 10,320 protein molecules were identified, of which 3,222 proteins were expressed in more than 90% of the samples. The Wilcox test was used to analyze differentially expressed proteins between paired tumor and normal tissue samples, as well as differentially expressed proteins in tumor tissue between the metastatic and non-metastatic groups, identifying a total of 12 differentially expressed proteins. Six differentially expressed proteins were upregulated in tumor tissues compared to normal tissues, and also upregulated in the metastatic group compared to the non-metastatic group. The remaining six differentially expressed proteins were downregulated in tumor tissues compared to normal tissues, and also downregulated in the metastatic group compared to the non-metastatic group. Finally, using the LASSO logistic algorithm and SVM logistic model, we selected the following four proteins—GGA1, FHL3, TGFBI, and NDUFS7—from the 12 differentially expressed proteins for further research and model building.

[0020] Example 2 This embodiment provides a method for constructing a prediction model for the risk of distant metastasis after surgery in colorectal cancer patients, and uses the model to predict the risk of distant metastasis after surgery in 193 colorectal cancer patients.

[0021] This embodiment describes a method for constructing a prediction model for the risk of distant metastasis after surgery in colorectal cancer patients. The method includes the following steps: detecting the expression level of the differentially expressed protein obtained in Example 1, obtaining the percentage (%) of FHL3+ tumor cells, GGA1+NUDFS7+ tumor cells, and TGFBI+ tumor stromal cells expressing the differentially expressed protein in the tissue, and substituting the percentage of each cell type into the calculation formula: Risk score = 1.500 × (Percentage of FHL3+ tumor cells) - 2.552 × (Percentage of GGA1+NUDFS7+ tumor cells) + 1.704 × (Percentage of TGFBI+ tumor stromal cells) . The risk score is used to determine the risk of distant metastasis after surgery in colorectal cancer patients. The threshold for the risk score is 0.978. If the risk score is ≤0.978, the sample is considered to have a low risk of distant metastasis after surgery; if it is >0.978, the sample is considered to have a high risk of distant metastasis after surgery. The above model was used to predict the risk of distant metastasis in 193 patients. The results are shown in Table 1 and... Figure 1 As shown in the figure, 91 patients were divided into a low-risk group and 102 patients were divided into a high-risk group. The predicted groupings and results are shown in Table 1 and 2. Figure 1 , Figure 2 As shown in Table 1 and Figure 1 It can be seen that, in the training set, internal validation set, and external validation set, the distant metastasis-free survival rate of patients in the high-risk group is significantly higher than that of patients in the low-risk group. Figure 1 Both the disease-free survival rate and the survival rate (Table 1) were poor.

[0022] Table 1

[0023] Example 3 In this embodiment, two validation groups were used to validate the postoperative distant metastasis risk prediction model for colorectal cancer patients constructed in Example 2.

[0024] The 193 patients in the internal validation group were divided into a high-risk group (107 cases) and a low-risk group (86 cases), as shown in Table 1 and Figure 1 , Figure 2 It can be seen that, compared with patients in the low-risk group, patients in the high-risk group had higher distant metastasis-free survival (HR 3.28, 95% CI 1.63-6.6; p<0.001) and disease-free survival (HR 2.16, 95% CI 1.27-3.67; p=0.008). Figure 1 Both B) and overall survival (Table 1) were poor.

[0025] The 316 patients in the external validation group were divided into a high-risk group (162 cases) and a low-risk group (154 cases), as shown in Table 1 and Figure 1 , Figure 2It can be seen that, compared with patients in the low-risk group, patients in the high-risk group had higher distant metastasis-free survival (HR 3.20, 95% CI 2.00-5.13; p<0.001) and disease-free survival (HR 1.62, 95% CI 1.11-2.36; p=0.014). Figure 1 Both C) and overall survival (Table 1) were poor.

[0026] Example 4 This embodiment uses ROC curves to evaluate the sensitivity and specificity of the proposed model for predicting the risk of distant metastasis after surgery in colorectal cancer patients. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the area under the ROC curve (AUC) of this prediction model is the highest for predicting the risk of distant metastasis after surgery in colorectal cancer patients, indicating that the prediction efficacy of this model for predicting the risk of distant metastasis after surgery in colorectal cancer patients is superior to other existing prediction methods.

[0027] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A set of biomarkers for predicting the risk of distant metastasis after surgery in patients with colorectal cancer, characterized in that, The biomarkers consist of FHL3 protein, GGA1 protein, NUDFS7 protein, and TGFBI protein; the colorectal cancer is stage II or stage III colorectal cancer.

2. The use of the biomarker for predicting the risk of postoperative distant metastasis in colorectal cancer patients according to claim 1 in the preparation of a reagent for predicting the risk of postoperative distant metastasis in colorectal cancer patients; wherein the colorectal cancer is stage II or stage III colorectal cancer.

3. The application according to claim 2, characterized in that, The reagents include those for detecting the expression levels of FHL3, GGA1, NUDFS7, and TGFBI proteins in tumor tissues using mass spectrometry-based proteomics technology, multiplex immunohistochemistry technology combined with artificial intelligence-based automated quantitative technology.

4. A method for constructing a predictive model for the risk of distant metastasis after surgery in patients with colorectal cancer, characterized in that, Includes the following steps: (1) Obtaining differentially expressed proteins: Collect the samples to be tested, extract the whole protein from the tissue, and use mass spectrometry analysis, Wilcox test analysis, LASSO logistic algorithm and SVM logistic model to analyze and screen the extracted whole protein to obtain differentially expressed proteins; (2) Constructing a prediction model for the risk of distant metastasis after surgery in colorectal cancer patients based on differentially expressed proteins: The expression level of the differentially expressed proteins obtained in step (1) was detected to obtain the composition ratio of tumor cells expressing the differentially expressed proteins in the tissue, and the composition ratio of each cell was substituted into the calculation formula to obtain the risk score; the risk of distant metastasis after surgery in colorectal cancer patients was judged based on the risk score; the differentially expressed proteins include FHL3 protein, GGA1 protein, NUDFS7 protein and TGFBI protein; the calculation formula is: risk score = 1.500 × FHL3 + The percentage of tumor cells (%) was -2.552 × GGA1. + NDUFS7 + The percentage of tumor cells (%) + 1.704 × TGFBI + The percentage of tumor stromal cells (%); the threshold of the risk score is 0.

978. If the risk score is ≤0.978, the sample is judged to be at low risk of distant metastasis after surgery; if it is >0.978, the sample is judged to be at high risk of distant metastasis after surgery.

Images