Application of two dipeptides in the preparation of a diagnostic kit to differentiate between prostate cancer and benign prostatic hyperplasia.

Abstract

This invention discloses the application of two dipeptides in the preparation of a diagnostic kit for differentiating prostate cancer from benign prostatic hyperplasia (BPH). The invention reveals that the combination of the dipeptides H-Asp-Phe-OH (DF) and H-Leu-Ala-OH (LA) with prostate-specific antigen (PSA) has high diagnostic value in differentiating prostate cancer from BPH, with a high accuracy rate. The combined diagnostic effect of these three peptides is significantly better than that of PSA alone in differentiating between prostate cancer and BPH, showing promise for development into a diagnostic kit for differentiating prostate cancer from BPH.

Description

Technical Field

[0001] This invention belongs to the field of disease detection and diagnosis, and relates to the application of biomarkers in disease diagnosis, specifically the application of two dipeptides in the preparation of a diagnostic kit for differentiating between prostate cancer and benign prostatic hyperplasia. Background Technology

[0002] Prostate cancer (PCa) is the most common malignant tumor in men. In recent years, with population aging and changes in lifestyle, the incidence of prostate cancer in my country has shown a continuous upward trend. Because early symptoms of prostate cancer are not obvious, most prostate cancer patients in my country are diagnosed at a stage of local progression or distant metastasis, leading to a poor prognosis. Although prostate-specific antigen (PSA) has been widely used for the early diagnosis of prostate cancer, elevated plasma PSA levels can also be seen in non-tumor states such as benign prostatic hyperplasia (BPH). Due to its poor specificity, PSA often leads to overdiagnosis and overtreatment, failing to meet clinical needs. Therefore, finding other ideal biomarkers to combine with PSA for joint diagnosis, and improving the ability of PSA to differentiate between prostate cancer and benign prostatic hyperplasia, is key to promoting accurate prostate cancer screening.

[0003] Small peptide metabolites are a class of small molecule metabolites composed of ten or fewer amino acids linked by peptide bonds, with an average molecular weight of less than 1000. With the rapid development of modern biology and nutrition, in the 1960s, scientists discovered that in addition to amino acids, some peptides, the digestive products of proteins in the small intestine, can enter the intestinal mucosal cells intact. By the 1990s, Professor Zou Yuandong and many other scientists confirmed that peptides are the main form in which proteins are absorbed by the human body. Peptides possess various biological activities and are widely involved in physiological processes such as cell proliferation, anti-inflammation, immune regulation, and antioxidation. Many diseases can lead to changes in the quantity and structure of small peptides expressed in urine, and these changes are of great significance for disease diagnosis.

[0004] In recent years, with the rapid development of metabolomics and proteomics, it has been revealed that urinary metabolites can serve as potential biomarkers for tumor diagnosis, becoming an important approach to finding diagnostic biomarkers for diseases. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide the application of dipeptides H-Asp-Phe-OH (DF) and H-Leu-Ala-OH (LA) in the preparation of a diagnostic kit for differentiating between prostate cancer and benign prostatic hyperplasia.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] Application of dipeptides H-Asp-Phe-OH and H-Leu-Ala-OH in combination with prostate-specific antigen in the preparation of a diagnostic kit to differentiate between prostate cancer and benign prostatic hyperplasia.

[0008] Preferably, the kit is a urine test kit.

[0009] More preferably, the kit contains reagents for detecting H-Asp-Phe-OH, H-Leu-Ala-OH, and prostate-specific antigen.

[0010] Beneficial effects:

[0011] This invention has discovered that the combination of dipeptides H-Asp-Phe-OH (DF) and H-Leu-Ala-OH (LA) with prostate-specific antigen (PSA) has high diagnostic value in differentiating between prostate cancer and benign prostatic hyperplasia (BPH), with a high diagnostic accuracy. The combined diagnostic effect of the three is significantly better than that of PSA alone in differentiating between prostate cancer and BPH, and it has the potential to be developed into a diagnostic kit for differentiating between prostate cancer and BPH. Attached Figure Description

[0012] Figure 1 The difference in the levels of dipeptides DF and LA in the urine of patients with prostate cancer and benign prostatic hyperplasia;

[0013] Figure 2 To validate the ROC curves and diagnostic threshold (cutoff) of dipeptide DF and LA combined with PSA in Set 1 for diagnosing and differentiating patients with prostate cancer and benign prostatic hyperplasia; for comparison, ROC curves of PSA alone for diagnosing and differentiating patients with prostate cancer and benign prostatic hyperplasia are also provided.

[0014] Figure 3 To verify the accuracy of the combination of dipeptide DF and LA with PSA in diagnosing prostate cancer patients and benign prostatic hyperplasia patients in set 2. Detailed Implementation

[0015] The substantive content of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but this is not intended to limit the scope of protection of the present invention.

[0016] Example 1: Diagnostic efficacy in differentiating between prostate cancer and benign prostatic hyperplasia

[0017] I. Experimental Samples

[0018] Sixty-three prostate cancer (PCA) patients and 58 benign prostatic hyperplasia (BPH) patients from Zhongda Hospital Affiliated to Southeast University were randomly assigned to three groups: a test set, a validation set 1, and a validation set 2. The test set consisted of 14 PCA patients and 22 BPH patients; validation set 1 consisted of 24 PCA patients and 21 BPH patients; and validation set 2 consisted of 25 PCA patients and 15 BPH patients.

[0019] Inclusion criteria: Prostate cancer patients were those diagnosed by pathology and who had not received radiotherapy or chemotherapy; Benign prostatic hyperplasia (BPH) patients were those whose BPH lesions were confirmed as benign by biopsy.

[0020] Exclusion criteria: ① Not meeting the relevant diagnostic criteria for prostate cancer and benign prostatic hyperplasia; ② Having other prostate diseases, such as prostatitis; ③ Having other serious liver, kidney, heart, lung, and hematopoietic system diseases.

[0021] II. Experimental Methods

[0022] 1. Collection and storage of urine samples

[0023] The study used a urinary catheter to collect approximately 10–12 ml of the first fresh midstream urine from the subjects in the morning, which was then aliquoted and stored at -80°C for long-term storage.

[0024] 2. Determination of dipeptide DF and LA content

[0025] The contents of dipeptides DF and LA were determined using conventional methods in this field, specifically as follows: After derivatization of dipeptides DF and LA with EOTMBA reagent, the contents of DF and LA dipeptides in the urine of the subjects were detected using a Shimadzu LCMS-Ultra-High Performance Liquid Chromatography Triple Quadrupole Mass Spectrometer. The specific experimental steps were as follows: An appropriate amount of urine sample was precisely pipetted and mixed with the EOTMBA methanol derivatization reagent, and reacted at 60℃ for 1.5 h; detection was performed using a Shimadzu LCMS-8045 UHPLC-Ultra-High Performance Liquid Chromatography Triple Quadrupole Mass Spectrometer. The chromatographic column used was an ACQUITYUPLC HSS T3 column (2.1×100nm, 1.8μM); the column temperature was controlled at 40℃. The mobile phase system consisted of phase A (10mM, pH=3 ammonium formate aqueous solution) and phase B (pure acetonitrile), with a flow rate set to 0.35 mL / min. The autosampler was controlled at 10℃, and the injection volume was 1 μL. The elution gradient was set as follows: 0-1 min, 10% B; 1-2 min, 10-15% B; 2-4 min, 15-20% B; 4-5 min, 20% B; 5-12 min, 20-25% B; 12-16 min, 25% B; 16-18 min, 25%-30% B; 18-18.1 min, 100% B; 18.1-19 min, 100% B; finally, the gradient was returned to equilibrium for 3 min. After detection, a standard curve was plotted with the ratio of the concentration of the target peptide derivative to the concentration of the isotopically labeled peptide derivative as the ordinate and the peptide concentration as the abscissa, thus achieving the detection of dipeptide content in the sample.

[0026] 3. Determination of prostate-specific antigen (PSA) levels

[0027] The PSA content in urine was measured using a commercially available PSA test kit, following the kit's instructions.

[0028] 4. Data Processing Methods

[0029] The content of the target dipeptide was expressed as mean ± standard deviation, and the Student's t-test was used to compare the differences in metabolite content among different groups. The diagnostic efficacy of dipeptides DF and LA for prostate cancer and benign prostatic hyperplasia was evaluated by plotting receiver operating characteristic (ROC) curves and calculating the area under the curve (AUC) and its 95% confidence interval (CI). DeLong's test was used to compare whether there were differences in AUC among different models, in order to comprehensively evaluate the application value of urinary DF and LA combined with PSA as a biomarker for prostate cancer diagnosis.

[0030] The diagnostic evaluation indicators are calculated as follows:

[0031]

[0032] Sensitivity, also known as the true positive rate, is the probability that a person actually has the disease and is correctly diagnosed as having the disease by a diagnostic test. Sensitivity = [a / (a+c)] × 100%. Specificity, also known as the true negative rate, is the probability that a person actually does not have the disease and is correctly diagnosed as not having the disease by a diagnostic test. Specificity = [d / (b+d)] × 100%.

[0033] III. Experimental Results

[0034] 1. Differences in the levels of dipeptides DF and LA in the urine of patients with prostate cancer and benign prostatic hyperplasia.

[0035] In the test set, compared with patients with benign prostatic hyperplasia, patients with prostate cancer showed significantly higher levels of DF and LA dipeptides in their urine. The measurement results are shown in the table below. Figure 1 As shown.

[0036]

[0037] 2. The efficacy of dipeptide DF and LA combined with PSA in diagnosing and differentiating prostate cancer from benign prostatic hyperplasia.

[0038] In validation set 1, the levels of dipeptides DF and LA, as well as PSA, in the urine of each patient were measured. ROC curves were plotted, and the area under the curve (AUC) and its 95% CI were calculated to evaluate the diagnostic efficacy of dipeptide DF and LA combined with PSA in differentiating between prostate cancer and benign prostatic hyperplasia. The ROC analysis results are as follows: Figure 2 As shown in the figure. In validation set 1, the AUC value of urinary DF and LA dipeptide combined with PSA in differentiating between prostate cancer and benign prostatic hyperplasia (BPH) was as high as 0.932 (sensitivity 0.929, specificity 0.818), with an optimal cutoff value of 0.263. The AUC value of PSA alone in differentiating between prostate cancer and BPH was only 0.86. Those skilled in the art know that an AUC of 0.5 indicates no diagnostic significance; an AUC of 0.5-0.7 indicates low diagnostic accuracy; an AUC of 0.7-0.9 indicates moderate diagnostic accuracy; and an AUC greater than 0.9 indicates high diagnostic accuracy. These experimental results indicate that the diagnostic efficacy of DF and LA dipeptide combined with PSA in differentiating between prostate cancer and BPH is significantly better than that of PSA alone.

[0039] 5. The accuracy of diagnosing and differentiating prostate cancer from benign prostatic hyperplasia by combining urinary dipeptide DF and LA with PSA.

[0040] In validation set 2, the levels of dipeptides DF, LA, and PSA in the urine of each patient were measured. The cutoff value of 0.263 obtained from validation set 1 was used as the diagnostic threshold; values ​​below this threshold were predicted as benign prostatic hyperplasia (BPH), and values ​​above this threshold were predicted as prostate cancer. The accuracy of the target dipeptides DF and LA combined with PSA in differentiating between prostate cancer and BPH was calculated by dividing the number of correctly predicted samples by the total number of samples. The results are as follows: Figure 3 As shown in validation set 2, the accuracy of diagnosing prostate cancer and benign prostatic hyperplasia patients by combining urinary dipeptide DF and LA with PSA was 77.5% (31 / 40), close to 80%, which is a high accuracy rate and has considerable potential for development and application in the field of prostate cancer diagnosis.

[0041] In summary, the combination of dipeptide DF and LA with PSA has high diagnostic value in differentiating between prostate cancer and benign prostatic hyperplasia (BPH), with a high diagnostic accuracy. The combined diagnostic effect of the three is significantly better than that of PSA alone in differentiating between prostate cancer and BPH, and it has the potential to be developed into a diagnostic kit for differentiating between prostate cancer and BPH.

[0042] Example 2: Diagnostic kit for differentiating between prostate cancer and benign prostatic hyperplasia

[0043] A urine test kit for diagnosing and differentiating between prostate cancer and benign prostatic hyperplasia, containing reagents for detecting DF and LA dipeptides and prostate-specific antigen.

[0044] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. Use of the dipeptides H-Asp-Phe-OH and H-Leu-Ala-OH in combination with prostate specific antigen for the preparation of a urine test kit for the diagnosis of prostate cancer versus benign prostatic hyperplasia.

2. Use according to claim 1, characterized in that: The kit comprises reagents for the detection of H-Asp-Phe-OH, H-Leu-Ala-OH and prostate specific antigen.

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