Application of Hsa-miR-24-3p in construction of pancreatic ductal adenocarcinoma postoperative total lifetime prediction model

Abstract

The invention discloses an application of Hsa-miR-24-3p in construction of a pancreatic ductal adenocarcinoma postoperative total lifetime prediction model. According to the invention, the expression of Hsa-miR-24-3p in preoperative serum of a PDAC patient is analyzed, and the total lifetime of a patient with increased level of Hsa-miR-24-3p (miR-24) is obviously reduced (Plt; 0.0001) of the method for manufacturing the same; multi-factor analysis shows that miR-24 is a prognosis factor independent of the age, gender and clinical pathological characteristics of a patient, and the prediction efficiency of miR-24 is remarkable; the area under the ROC curve reaches 0.82, which is superior to the area under the CA19-9 (0.61), the area under the CA125 (0.59), the area under the CA50 (0.51) and the area under the CEA (0.56); when the miR-24 and the TNM are staging, and the CA19-9 and the CA125 are integrated into the column diagram model, the prognosis prediction accuracy is further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of prognostic models, and particularly relates to the application of Hsa-miR-24-3p in constructing a prediction model for the overall survival of patients after pancreatic ductal adenocarcinoma surgery. Background Art

[0002] Pancreatic cancer is a highly malignant tumor with extremely poor prognosis, and the pancreatic ductal adenocarcinoma (PDAC) subtype accounts for more than 90% of all pancreatic malignancies. Due to the lack of early diagnosis and effective treatment methods, pancreatic cancer has become the fourth leading cause of cancer-related deaths, with a 5-year relative survival rate of only 12%. Although surgical resection is currently the only treatment method with the potential for cure, approximately 80%-85% of pancreatic cancer patients have local invasion or metastasis at the time of diagnosis and are unable to undergo surgical resection. As the main treatment method for pancreatic cancer, conventional adjuvant chemotherapy faces significant drug resistance challenges. Even immunotherapy, which has shown good prospects in other cancer types, has not achieved a breakthrough in the treatment of pancreatic cancer due to the unique immunosuppressive tumor microenvironment of pancreatic cancer. It is worth noting that due to differences in individual genetic backgrounds and biological characteristics, the degree of benefit of patients from the same treatment regimen varies. Therefore, identifying prognostic markers that can effectively predict the treatment response of patients has important clinical significance.

[0003] Some molecular alterations have been confirmed to be promising biomarkers. Traditionally, serum carbohydrate antigens (CA) 19-9, CA125, CA50, and carcinoembryonic antigen (CEA) have been commonly used for the diagnosis, metastasis evaluation, and prognosis judgment of pancreatic cancer, but their efficacy is low. CA19-9 is currently the only biomarker approved by the US Food and Drug Administration for the diagnosis and prognosis judgment of pancreatic cancer. However, in a randomized controlled phase III clinical trial, the baseline CA19-9 level was not an independent survival predictor. In addition, multiple studies have reported the relationship between systemic inflammation markers (especially neutrophil-lymphocyte ratio [NLR], platelet-lymphocyte ratio [PLR], monocyte-lymphocyte ratio [MLR], and neutrophil-monocyte ratio [NMR]) and the overall survival of PDAC patients. However, no consensus has been reached on their prognostic value.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The object of the present invention is to provide an application of Hsa-miR-24-3p in constructing a prediction model for the overall survival after pancreatic ductal adenocarcinoma surgery. In this application, the expression of Hsa-miR-24-3p in the preoperative serum of PDAC patients was analyzed, and it was determined that the expression of Hsa-miR-24-3p is an independent marker for predicting overall survival, and thus can be used to construct a prediction model for the overall survival after pancreatic ductal adenocarcinoma surgery.

[0006] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

[0007] The first aspect of the present invention provides an application of Hsa-miR-24-3p in constructing a prediction model for the overall survival after pancreatic ductal adenocarcinoma surgery.

[0008] The second aspect of the present invention provides a prediction model for the overall survival after pancreatic ductal adenocarcinoma surgery. The prediction model for the overall survival after pancreatic ductal adenocarcinoma surgery predicts the overall survival of patients after pancreatic ductal adenocarcinoma surgery according to the delta Ct value of Hsa-miR-24-3p in the patient's serum.

[0009] Preferably, the prediction of the overall survival of patients after pancreatic ductal adenocarcinoma surgery according to the delta Ct value of Hsa-miR-24-3p in the patient's serum includes:

[0010] If the delta Ct value of Hsa-miR-24-3p in the patient's serum < -1.905, the patient is in the high-risk group; if the delta Ct value of Hsa-miR-24-3p in the patient's serum ≥ -1.905, the patient is in the low-risk group;

[0011] The overall survival of patients in the low-risk group is greater than that of patients in the high-risk group.

[0012] The third aspect of the present invention provides an application of Hsa-miR-24-3p combined with CA19-9, CA125 and TNM staging in constructing a prediction model for the overall survival after pancreatic ductal adenocarcinoma surgery.

[0013] The fourth aspect of the present invention provides a prediction model for the overall survival after pancreatic ductal adenocarcinoma surgery. The prediction model for the overall survival after pancreatic ductal adenocarcinoma surgery is a nomogram model, and the nomogram model is constructed based on Hsa-miR-24-3p, CA19-9, CA125 and TNM staging.

[0014] Preferably, the nomogram model consists of 9 scales. The first scale is a score scale, and the score range is 0 - 100;

[0015] The second scale is the TNM staging scale;

[0016] The third scale is the CA19-9 scale, and the CA19-9 concentration range is 0 to 1000;

[0017] The fourth scale is the CA125 scale, and the CA124 concentration range is 0 to 600;

[0018] The fifth scale is the miR24 scale. miR24 refers to the delta Ct value of Hsa-miR-24-3p, and the value range is 4 to -10;

[0019] The sixth scale is the total score, and the range is 1 to 120;

[0020] The seventh scale is the survival probability with an overall survival greater than 1 year, and the range is 0.9 to 0.1;

[0021] The eighth scale is the survival probability with an overall survival greater than 2 years, and the range is 0.8 to 0.1;

[0022] The ninth scale is the survival probability with an overall survival greater than 5 years, and the range is 0.7 to 0.1.

[0023] Compared with the prior art, the beneficial effects of the present invention at least include:

[0024] This application analyzed the expression of Hsa-miR-24-3p in the preoperative serum of PDAC patients, determined that the expression of Hsa-miR-24-3p is an independent marker for predicting the overall survival, and thus can be used to construct a prediction model for the overall survival after pancreatic ductal adenocarcinoma surgery. Specifically, the research results show that the overall survival of patients with elevated levels of Hsa-miR-24-3p (miR-24) is significantly lower than that of patients in the low-risk group (P<0.0001); multivariate analysis shows that miR-24 is a prognostic factor independent of patient age, gender, and clinicopathological characteristics, and its predictive efficacy is significant; the area under the ROC curve reaches 0.82, which is better than CA19-9 (0.61), CA125 (0.59), CA50 (0.51), and CEA (0.56); when miR-24, TNM stage, CA19-9, and CA125 are integrated into the nomogram model, the prognostic prediction accuracy is further improved. In addition, patients who received postoperative adjuvant chemotherapy in the high-risk group had better overall survival and two-year survival rates than those who did not receive adjuvant chemotherapy (P<0.0001); it can be seen that the prediction model for pancreatic ductal adenocarcinoma after surgery of the present invention can assist in the treatment of PDAC. Description of the Drawings

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0026] Figure 1 Baseline characteristics of patients in the embodiments of the present invention;

[0027] Figure 2 Analysis results of the prognostic value of miR-24 in PDAC patients in the embodiments of the present invention;

[0028] Figure 3 Research results of miR-24 in differentiating adjuvant chemotherapy for PDAC patients in the embodiments of the present invention;

[0029] Figure 4 Independent prognostic factors for pancreatic cancer in the embodiments of the present invention;

[0030] Figure 5 Effect of miR-24 on OS in different subgroups of PDAC patients in the embodiments of the present invention;

[0031] Figure 6 Survival analysis results of miR-24 in different patient subgroups in the embodiments of the present invention;

[0032] Figure 7 Predictive nomogram model for OS after pancreatic ductal adenocarcinoma surgery in the embodiments of the present invention and comparison results of the concordance index with various factors. Specific embodiments

[0033] The following will describe in detail the embodiments of the technical solutions of the present invention in combination with the embodiments. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, so they are only examples and cannot be used to limit the protection scope of the present invention.

[0034] It should be noted that unless otherwise specified, the technical terms or scientific terms used in this application should have the ordinary meanings understood by those skilled in the art to which the present invention belongs.

[0035] The embodiments of the present invention provide an application of Hsa-miR-24-3p in constructing a predictive model for the overall survival after pancreatic ductal adenocarcinoma surgery.

[0036] The present invention analyzed the expression of Hsa-miR-24-3p in the preoperative serum of PDAC patients. Specifically, the overall survival of patients with elevated levels of Hsa-miR-24-3p (miR-24) was significantly reduced compared to that of patients in the lower-risk group (P<0.0001); multivariate analysis showed that miR-24 was a prognostic factor independent of patient age, gender, and clinicopathological features, and its predictive efficacy was significant; the area under the ROC curve reached 0.82, which was superior to CA19-9 (0.61), CA125 (0.59), CA50 (0.51), and CEA (0.56). Therefore, it was determined that the expression of Hsa-miR-24-3p was an independent marker for predicting overall survival, and thus could be used to construct a predictive model for the overall survival of patients after pancreatic ductal adenocarcinoma surgery.

[0037] Another embodiment of the present invention provides a predictive model for the overall survival of patients after pancreatic ductal adenocarcinoma surgery. The predictive model for the overall survival of patients after pancreatic ductal adenocarcinoma surgery predicts the overall survival of patients after pancreatic ductal adenocarcinoma surgery based on the delta Ct value of Hsa-miR-24-3p in the patient's serum.

[0038] In one embodiment, predicting the overall survival of patients after pancreatic ductal adenocarcinoma surgery based on the delta Ct value of Hsa-miR-24-3p in the patient's serum includes:

[0039] If the delta Ct value of Hsa-miR-24-3p in the patient's serum < -1.905, the patient is in the high-risk group; if the delta Ct value of Hsa-miR-24-3p in the patient's serum ≥ -1.905, the patient is in the low-risk group;

[0040] The overall survival of patients in the low-risk group is greater than that of patients in the high-risk group.

[0041] Another embodiment of the present invention provides an application of Hsa-miR-24-3p combined with CA19-9, CA125, and TNM staging in constructing a predictive model for the overall survival of patients after pancreatic ductal adenocarcinoma surgery.

[0042] When miR-24, TNM staging, CA19-9, and CA125 are integrated into a nomogram model, the prognostic prediction accuracy is further improved.

[0043] Another embodiment of the present invention provides a predictive model for the overall survival of patients after pancreatic ductal adenocarcinoma surgery. The predictive model for the overall survival of patients after pancreatic ductal adenocarcinoma surgery is a nomogram model, and the nomogram model is constructed based on Hsa-miR-24-3p, CA19-9, CA125, and TNM staging.

[0044] In one embodiment, the nomogram model consists of nine scales. The first scale is a score scale with a score range of 0 to 100;

[0045] The second scale is the TNM staging scale;

[0046] The third scale is the CA19-9 scale, with a CA19-9 concentration range of 0 to 1000;

[0047] The fourth scale is the CA125 scale, with a CA124 concentration range of 0 to 600;

[0048] The fifth scale is the miR24 scale, where miR24 refers to the delta Ct value of Hsa-miR-24-3p, with a value range of 4 to -10;

[0049] The sixth scale is the total score, with a range of 1 to 120;

[0050] The seventh scale is the survival probability of overall survival greater than 1 year, with a range of 0.9 to 0.1;

[0051] The eighth scale is the survival probability of overall survival greater than 2 years, with a range of 0.8 to 0.1;

[0052] The ninth scale is the survival probability of overall survival greater than 5 years, with a range of 0.7 to 0.1.

[0053] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0054] Embodiment

[0055] A retrospective analysis was performed on 296 patients with stage I-IV PDAC who underwent radical resection at Renji Hospital Affiliated to Shanghai Jiao Tong University from October 2012 to July 2019. The patient characteristics are shown in detail in Figure 1 . A total of 177 males (59.8%) and 119 females (40.2%) were included in the study. The median age of the patients was 66 years (range 24 - 89 years). Patients were screened according to the following exclusion criteria: 1) having other primary tumors simultaneously; 2) receiving preoperative treatment; 3) lacking baseline or follow-up data; 4) dying from non-pancreatic cancer-related causes, such as complications, other malignant diseases, or accidents. Patients were followed up every three months until death or the end of the follow-up. The overall survival (OS) was defined as the time (in months) from the surgery date to the death date or the last follow-up.

[0056] This study was approved by the Ethics Committee of Dalian University of Technology (reference number: 2020-075), and informed consent was obtained from all patients. PDAC staging was performed according to the TNM staging system (8th edition of AJCC). The study was carried out in accordance with the principles of the Declaration of Helsinki regarding research involving human subjects. All experiments were conducted in accordance with the recommendations for reporting prognostic studies of tumor markers.

[0057] Real-time polymerase chain reaction (RT-PCR) detection of serum miR-24:

[0058] The serum miR-24 level was determined by RT-PCR using TaqMan probes. Total RNA was extracted from 0.25 mL of serum samples using RNAiso Blood (Takara, Japan) according to the manufacturer's instructions. Subsequently, the RNA was transcribed into cDNA in a reverse transcription reaction with a reduced volume (10 μL). Then, 45 cycles of amplification RT-PCR were performed on a LightCycler 480II system (Roche, Switzerland). The serum level of small nuclear RNA U6 was measured simultaneously as an internal reference control. Each sample was measured in triplicate, and the Ct value of miR-24 or U6 was calculated as the average of the triplicate values. The endogenous level of miR-24 in serum was calculated using the delta Ct value, that is, the normalized Ct value of miR-24 relative to U6.

[0059] Statistical analysis

[0060] Continuous variables were compared using Student's t-test or one-way analysis of variance, and categorical variables were tested using the chi-square test. Receiver operating characteristic (ROC) curve analysis was used to evaluate the prognostic efficacy of variables. The endogenous level of miRNA expression was calculated using the delta Ct method (ΔCtsample = CtmiRNA - CtU6). Patients were divided into high-risk and low-risk groups according to the miR-24 level. Kaplan-Meier curve analysis was used to analyze the difference in OS and the Log rank test was used for comparison. Univariate Cox proportional hazards regression analysis was performed on all variables, and variables with P < 0.05 were included in the multivariate Cox proportional hazards regression analysis to determine independent prognostic factors. In addition, a nomogram model was established based on risk factors to predict the 1-year, 2-year, and 5-year risks and OS of PDAC patients. The C-index was used to evaluate the accuracy of the nomogram, and the calibration curve was used to verify the discrimination of the nomogram. Statistical analysis was performed using R Studio (version 4.3.1) or GraphPad Prism 9 software. P < 0.05 was considered statistically significant;

[0061] 1. The research results on the prognostic value of miR-24 in PDAC patients are as Figure 2 shown;

[0062] Figure 2 Expression levels of miR-24 in sera of (A) surviving and deceased patients with PDAC. (B) ROC analysis of miR-24 in surviving and deceased patients with PDAC. (C) Kaplan-Meier survival curves of patients in high-risk and low-risk groups based on miR-24. (D) Survival status of patients in high-risk and low-risk groups based on miR-24. (E) Proportion of surviving and deceased patients showing low or high levels of miR-24. (F) Mean survival time of PDAC patients with low or high miR-24 levels. Data are presented as mean ± SEM. (G) OS and 2-year dependent ROC curves of miR-24 and serum protein markers. (H) OS and 2-year dependent ROC curves of miR-24 and systemic inflammation markers. NLR, neutrophil-lymphocyte ratio. PLR, platelet-lymphocyte ratio. MLR, monocyte-lymphocyte ratio. NMR, neutrophil-monocyte ratio;

[0063] As shown in Figure 2 A, the delta Ct value of miR-24 in the deceased patient group was significantly lower than that in the surviving patient group, indicating an increased expression level of miR-24 in deceased patients (P < 0.0001). Subsequently, ROC analysis was performed to evaluate the efficacy of miR-24 in predicting OS, and the area under the curve reached 0.82 (95% CI, 0.78 - 0.88; Figure 2 B). Based on the ROC analysis, the optimal cut-off value of miR-24 was determined to be -1.905, and patients were divided into a high-risk group (delta Ct < -1.905) and a low-risk group (delta Ct ≥ -1.905). The OS of patients in the high-risk group was significantly lower than that in the low-risk group (P < 0.0001; Figure 2 C). Waterfall plot analysis showed that the proportion of deceased patients with elevated miR-24 levels was significantly higher than that of patients with decreased miR-24 levels ( Figure 2 D, E). The median survival time of patients with elevated miR-24 levels was 14.5 months, which was significantly shorter than that of patients with low miR-24 levels (P < 0.001; Figure 2 F). These data indicate that miR-24 has good prognostic predictive value in PDAC patients.

[0064] Through time-dependent ROC analysis, miR-24 showed stronger predictive ability than traditional markers, with a significantly larger area under the curve; compared with serum protein markers CA19-9, CA125, CA50, and CEA, the areas under the curve of miR-24 in predicting OS and 2-year survival reached 0.82 and 0.73, respectively ( Figure 2In view of the prognostic potential of systemic inflammatory markers in routine blood tests reported in some studies, the predictive abilities of NLR, PLR, MLR, and NMR were evaluated. The results showed that these inflammatory markers failed to effectively predict the OS or 2-year survival of PDAC patients ( Figure 2 In part H). These comparison results further confirmed that miR-24 can be used as a prognostic biomarker for PDAC.

[0065] 2. Study of miR-24 in differentiating chemotherapy for PDAC patients:

[0066] To develop effective biomarkers for clinical application, it is necessary to explore the response of miR-24 to ACT. The results are as Figure 3 shown, Figure 3 Kaplan-Meier curves of OS and 2-year survival rate in all patients (A, B), the high-risk group defined by miR-24 (C, D), and the low-risk group defined by miR-24 (E, F);

[0067] As Figure 3 known, in the overall patient cohort, it was observed that although postoperative ACT did not improve OS, the 2-year survival rate was increased due to ACT ( Figure 3 in A, B of part). This result may be explained by the fact that patients benefit from short-term chemotherapy while also developing long-term drug resistance. Interestingly, in the high-risk group defined by miR-24, both OS and 2-year survival rate were significantly improved after receiving ACT (P < 0.0001; Figure 3 in C, D of part). In contrast, in the low-risk group, neither OS nor 2-year survival rate benefited from ACT (P > 0.05; Figure 3 in E, F of part). These findings highlight the potential of miR-24-based individualized prognostic prediction, which can assist in the treatment of PDAC, effectively reduce unnecessary medical interventions, and reduce the economic burden on patients.

[0068] 3. Confirmation that miR-24 is an independent prognostic factor for PDAC:

[0069] By performing univariate and multivariate Cox regression analyses on demographic and clinicopathological parameters, independent prognostic factors for PDAC were determined. The results of univariate Cox regression analysis showed that TNM stage, CA19-9, CA125, and miR-24 are promising prognostic factors for PDAC (as Figure 4)。However, the data from the multivariate Cox regression analysis confirmed that only miR-24 and TNM stage IV were independent prognostic factors for PDAC (P < 0.0001 and P = 0.016, respectively). As is well known, TNM stage IV refers to cases of distant metastasis, and their survival status is generally poor. Therefore, stage IV is widely recognized as an independent factor affecting the prognosis of pancreatic cancer patients. In addition, miR-24 showed the effectiveness in predicting OS in most subgroups stratified by demographic and clinicopathological parameters, including gender, age, BMI, smoking, alcohol consumption, hypertension, diabetes, tumor location, T stage, N stage, and histological grade ( Figure 5 , and the results were tested by Cox proportional hazards regression analysis). These exciting findings were further supported by the Kaplan-Meier analysis of subgroups based on T stage, N stage, histological grade, and tumor location ( Figure 6 ). In summary, miR-24 can serve as an independent prognostic indicator for PDAC patients.

[0070] 4. Nomogram model based on miR-24 and clinicopathological factors:

[0071] Given the significant prognostic value of miR-24 and the factors screened by univariate Cox regression analysis, a nomogram model integrating multiple variables was constructed. Figure 7 In A, it is a predictive nomogram model for predicting the OS of pancreatic cancer patients, and the top scale corresponds to the variable axis. The sum of these scores is on the total score axis. By drawing a vertical line downward from the total score axis to the survival axis, the overall survival probabilities of patients at 1 year, 2 years, and 5 years can be predicted. Based on this model, individualized survival prediction can be performed through the TNM stage and the levels of serum CA19-9, CA125, and miR-24 of patients. The predictive ability of the model was verified using the concordance index and calibration curve. The concordance index of the nomogram was significantly higher than that of its individual component factors, including miR-24 ( Figure 7 In B). From the 1-year calibration curve, there was a significant correlation between the actual data and the model predicted values, but this consistency decreased with the extension of survival time ( Figure 7 In C-E). In summary, this model has good efficacy in predicting the prognosis of patients.

[0072] 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 them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered by the scope of the claims and the description of the present invention.

Claims

1. Application of Hsa-miR-24-3p in constructing a prediction model for overall survival after surgery for pancreatic ductal adenocarcinoma.

2. A model for predicting overall survival after surgery for pancreatic ductal adenocarcinoma, characterized in that: The overall survival prediction model for pancreatic ductal adenocarcinoma after surgery predicts the overall survival of patients after pancreatic ductal adenocarcinoma after surgery based on the delta Ct value of Hsa-miR-24-3p in the patient's serum.

3. The overall survival prediction model for pancreatic ductal adenocarcinoma after surgery according to claim 2, characterized in that: The method of predicting the overall survival of patients after pancreatic ductal adenocarcinoma surgery based on the delta Ct value of Hsa-miR-24-3p in the patient's serum includes: If the delta Ct value of Hsa-miR-24-3p in the patient's serum is <-1.905, the patient is in the high-risk group, and if the delta Ct value of Hsa-miR-24-3p in the patient's serum is ≥-1.905, the patient is in the low-risk group; The overall survival of patients in the low-risk group is greater than that of patients in the high-risk group.

4. Application of Hsa-miR-24-3p combined with CA19-9, CA125 and TNM staging in constructing a prediction model for overall survival after surgery for pancreatic ductal adenocarcinoma.

5. A model for predicting overall survival after surgery for pancreatic ductal adenocarcinoma, characterized in that: The overall survival prediction model for pancreatic ductal adenocarcinoma after surgery is a nomogram model, which is constructed based on Hsa-miR-24-3p, CA19-9, CA125 and TNM staging.

6. The overall survival prediction model for pancreatic ductal adenocarcinoma after surgery according to claim 5, characterized in that: The nomogram model is composed of 9 scales, the first scale is a score scale, and the score range is 0 to 100; The second scale is the TNM staging scale; The third scale is the CA19-9 scale, and the CA19-9 concentration range is 0 to 1000; The fourth scale is the CA125 scale, and the CA124 concentration range is 0 to 600; The fifth ruler is the miR24 ruler, where miR24 refers to the delta Ct value of Hsa-miR-24-3p, ranging from 4 to −10; The sixth scale is the total score, ranging from 1 to 120; The seventh scale is the probability of survival with an overall survival greater than 1 year, ranging from 0.9 to 0.1; The eighth scale is the probability of survival with an overall survival of more than 2 years, ranging from 0.8 to 0.1; The ninth scale is the probability of overall survival greater than 5 years, ranging from 0.7 to 0.1.

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