Application of myristic acid as marker in diagnosis of prostatic cancer

Abstract

The invention discloses an application of myristic acid as a marker in diagnosis of prostate cancer, and particularly provides myristic acid as a biomarker for screening prostate cancer, the myristic acid has higher specificity and better diagnosis efficiency than TPSA, can reduce unnecessary prostate biopsy puncture and alleviate pain of patients, and can be used as a biomarker for screening prostate cancer. And missed diagnosis of conventional puncture is reduced.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to an application of myristic acid as a marker in diagnosing prostate cancer. Background Art

[0002] Prostate cancer (PCa) is one of the most common cancers worldwide and accounts for a large proportion of all cancer-related deaths. Early screening is crucial for the treatment and prognosis of prostate cancer (PCa). Currently, the total prostate-specific antigen (TPSA) test in serum is one of the most commonly used methods for screening prostate cancer (PCa), with a critical value of 4 ng / mL. When the TPSA level is >4 ng / mL, it indicates that the patient has a higher risk of prostate cancer (PCa), and when the TPSA level is >10 ng / mL, it is highly suspected of having prostate cancer (PCa). However, some benign prostate diseases such as prostatic hyperplasia and prostatitis can also cause TPSA levels to increase and be greater than 4 ng / mL. Screening for prostate cancer (PCa) based on TPSA levels >4 ng / mL will result in a high false positive rate and poor specificity, resulting in unnecessary prostate cancer biopsies and increasing physical and psychological pain for patients.

[0003] Patients with TPSA levels >4ng / mL often undergo biopsy to confirm prostate cancer (PCa). However, studies have shown that the standard prostate cancer (PCa) biopsy method will miss 77.7% of prostate cancer (PCa) patients with prostate cancer located in the anterior prostate and transition zone. Therefore, there is an urgent need for highly specific biomarkers to assess the risk of prostate cancer (PCa) in patients with TPSA>4ng / mL. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a biomarker for distinguishing prostate cancer from benign prostatic hyperplasia, the biomarker is phosphatidylethanolamine (20:0 / 15:0) (PE (20:0 / 15:0)) or myristic acid, both of which can distinguish prostate cancer from benign prostatic hyperplasia.

[0005] The purpose of the present invention is achieved through the following technical solutions.

[0006] Application of PE (20:0 / 15:0) as a marker in the preparation of products for diagnosing prostate cancer.

[0007] In the above technical solution, the premise for using the product is that the TPSA level in the serum of the patient detected by the product is >4ng / mL.

[0008] A product for diagnosing prostate cancer, which detects PE (20:0 / 15:0) in patient plasma and uses PE (20:0 / 15:0) as a marker for diagnosing prostate cancer.

[0009] In the above technical solution, the premise for using the product is that the TPSA level in the serum of the patient detected by the product is >4ng / mL.

[0010] Use of myristic acid as a marker in the preparation of products for diagnosing prostate cancer.

[0011] In the above technical solution, the premise for using the product is that the TPSA level in the serum of the patient detected by the product is >4ng / mL.

[0012] A product for diagnosing prostate cancer, which detects myristic acid in a patient's plasma and uses myristic acid as a marker for diagnosing prostate cancer.

[0013] In the above technical solution, the premise for using the product is that the TPSA level in the serum of the patient detected by the product is >4ng / mL.

[0014] The PE (20:0 / 15:0) and myristic acid provided by the present invention can both be used as biomarkers for relatively accurate screening of prostate cancer. The PE (20:0 / 15:0) and myristic acid as biomarkers have higher specificity and better diagnostic efficacy than TPSA, can reduce unnecessary prostate biopsy punctures, alleviate patient pain, and reduce missed diagnoses in conventional punctures. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Orthogonal partial least squares discriminant analysis diagram of BEH C18 group, HSS T3 group and CSH PhenylHexyl group;

[0016] Figure 2 The volcano plots are for BEH C18, HSS T3, and CSH PhenylHexyl;

[0017] Figure 3 Receiver operating characteristic curves for PE (20:0 / 15:0) and TPSA (10 ng / mL) to differentiate patients with prostate cancer and prostate hyperplasia with TPSA>4 ng / ml;

[0018] Figure 4 Receiver operating characteristic curves for myristic acid and TPSA (10 ng / mL) for differentiating patients with prostate cancer and prostate hyperplasia with TPSA>4 ng / ml. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0020] Ultra-performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry (UPLC-IMS-QToF), machine model: Vion-IMS-QTOF;

[0021] ACQUITY BEH C18 chromatographic column, the diameter of the column filler particles is 1.7 μm, the inner diameter × length is 2.1 × 100 mm, purchased from Waters, Ireland;

[0022] ACQUITY HSS T3 chromatographic column, the diameter of the column filler particles is 1.8 μm, the inner diameter × length is 2.1 × 100 mm, purchased from Waters, Ireland;

[0023] ACQUITY Premier CSH Phenyl-Hexyl Column with VanGuard FIT chromatographic column, the diameter of the chromatographic column filler particles is 1.7 μm, the inner diameter × length is 2.1 × 100 mm, purchased from Waters, Ireland;

[0024] Progenesis QI v3.0 software, Waters, Nonlinear Dynamics, Newcastle, UK;

[0025] SIMCA 14.1 software, Umetrics AB, Sweden.

[0026] In the following examples, plasma samples were collected from 90 participants recruited from the Department of Urology, The Affiliated Hospital of North China University of Technology, Tangshan, Hebei Province between June 2023 and February 2024, of which 50 participants with benign prostatic hyperplasia (BPH) were used as the prostate hyperplasia group, and 40 participants with prostate cancer (PCa) were used as the prostate cancer group.

[0027] The inclusion criteria for all participants in this invention include:

[0028] (1) All participants must be between 35 and 80 years old;

[0029] (2) TPSA in serum of all participants was >4 ng / mL;

[0030] (3) The participants in the prostate cancer group had undergone transrectal prostate tissue puncture biopsy and were initially diagnosed with prostate cancer, and the participants had not yet received any related treatments such as endocrine therapy, radiotherapy, chemotherapy, and surgery;

[0031] (4) Participants in the prostatic hyperplasia group were diagnosed with benign prostatic hyperplasia by imaging and accompanied by symptoms of lower urinary tract obstruction (such as frequent urination, urgency, and slow urine flow) or were finally diagnosed with benign prostatic hyperplasia by prostate puncture biopsy;

[0032] (5) The presence of other related diseases, especially blood system diseases, connective tissue diseases, severe cardiovascular and cerebrovascular diseases, and other serious urinary system diseases, were excluded from both the prostate hyperplasia group and the prostate cancer group.

[0033] In the following examples, each plasma sample is venous blood from a participant one day after admission. To eliminate the influence of factors such as diet, the participants fasted for at least 8 hours overnight, and EDTA anticoagulated blood samples were collected between 6 and 10 a.m. After multiple centrifugation, plasma samples were separated and quickly stored in a -80°C refrigerator for subsequent processing. The plasma samples were completely thawed at room temperature before use.

[0034] The study protocol was approved by the Ethics Committee of the Affiliated Hospital of North China University of Technology and was conducted in accordance with the principles set forth in the Declaration of Helsinki. All participants provided written consent prior to enrollment. The comprehensive clinical and pathological data of the participants are shown in Table 1.

[0035] Table 1

[0036]

[0037]

[0038] Note: Data is based on express, represents the mean, s represents the standard deviation, * represents P < 0.05; ** represents P < 0.01; *** The representative value is P < 0.001. Gleason score is the Gleason score of prostate cancer, which is expressed as the score of the main growth pattern + the score of the secondary growth pattern.

[0039] Example 1

[0040] Example 1-1 Extraction of plasma metabolites

[0041] Each plasma sample (0.1 mL) was placed in a 2 mL centrifuge tube, and 0.3 mL of methanol-acetonitrile mixture (methanol-acetonitrile mixture is a mixture of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 1:1) pre-cooled in a 0°C ice bath was added and mixed on dry ice. The centrifuge tube was fully vortexed for 30 seconds using a fast mixer, and incubated at -20°C for 2 hours to precipitate the protein. After incubation, it was ultrasonically treated for 5 minutes, centrifuged at 15000 rpm and 4°C for 10 minutes, the supernatant was aspirated and centrifuged again for 10 minutes, 200 uL of the supernatant was aspirated and filtered with a nylon filter (membrane pore size: 0.2 μm), and plasma metabolites were obtained after filtration. The plasma metabolites were transferred to an injection bottle with an inner cannula (Waters, Elstree, UK) for storage.

[0042] Example 1-2 Plasma metabolomics screening for markers that can distinguish between prostate cancer and benign prostatic hyperplasia with TPSA>4 ng / mL

[0043] Plasma metabolites were separated and detected by ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry (UPLC-IMS-QToF): In order to improve the resolution, three chromatographic columns were used to separate plasma metabolites. The three chromatographic columns were ACQUITY BEH C18 column, ACQUITY column with good separation effect for polar metabolites HSS T3 chromatographic column and ACQUITY Premier CSH Phenyl-Hexyl Column with VanGuardTM FIT chromatographic column with ion exchange function. ACQUITY The separation conditions of the BEH C18 column are as follows: the mobile phase is a mixture of mobile phase A and mobile phase B, mobile phase A is 0.1% (v / v) formic acid in water, mobile phase B is methanol containing 0.1% (v / v) formic acid, the gradient is: 0min-1min-2.5min-6.5min-7min-12min, the volume fraction of mobile phase B in the mobile phase changes with the gradient: 1%-30%-60%-90%-100%-100%, the flow rate is 0.4mL / min, the column temperature is 50°C, and the injection volume of plasma metabolites is 2μL. ACQUITY The separation conditions of the HSS T3 column are as follows: the mobile phase is a mixture of mobile phase A and mobile phase B, mobile phase A is 0.1% (v / v) formic acid aqueous solution, and mobile phase B is acetonitrile containing 0.1% (v / v) formic acid. The gradient is: 0min-3min-16min, the volume fraction of mobile phase B in the mobile phase changes with the gradient: 3%-3%-100%, the flow rate is 0.4mL / min, the column temperature is 40°C, and the injection volume of plasma metabolites is 4μL. The separation conditions of ACQUITY Premier CSH Phenyl-Hexyl Column with VanGuardTM FIT chromatographic column are as follows: the mobile phase is a mixture of mobile phase A and mobile phase B, mobile phase A is 0.1% (v / v) formic acid aqueous solution, mobile phase B is acetonitrile containing 0.1% (v / v) formic acid, the gradient is: 0min-4min-7min-8min, the volume fraction of mobile phase B in the mobile phase changes with the gradient: 1%-25%-95%-95%, the flow rate is 0.4mL / min, the column temperature is 45℃, and the injection volume of plasma metabolites is 4μL. The storage temperature of plasma metabolites is kept at 4℃.

[0044] After separation by chromatographic column, plasma metabolites were directly separated by ion mobility in positive ion mode (ESI+) and negative ion mode (ESI-), and then entered mass spectrometry detection through ESI source: After separation, High Definition MS E The modes were used to harvest mass spectrometry data of plasma metabolites separated in two different ion modes (positive ion mode and negative ion mode), and the mass spectrometry parameters were set as follows: the capillary voltages were 2.4 kV (ESI-) and 3.2 kV (ESI+), the mass-to-charge ratio (m / z) range of the metabolites to be measured was 50-1000 Da, the source temperature was 120 °C, the desolvation temperature was 350 °C, the desolvation gas flow rate was 1000 L / h, the conical gas flow rate was 50 L / h, and the mass-to-charge ratio of leucine enkephalin was 556.2766 m / z (ESI+) and 554.2620 m / z (ESI-) for lock mass calibration. The interval time was 0.5 min, the sampling time was 0.5 min, the collision energy (CE) was 18 eV, and the flow rate was 10 μL / min.

[0045] Example 1-3 Data Processing and Analysis

[0046] The mass spectrometry data of plasma metabolites separated by each chromatographic column and then further separated in positive and negative ion modes were collected by Waters UNIFI 1.8.1 software. After lock mass calibration, the data were imported into ProgenesisQI v3.0 for differential metabolite analysis. Peak extraction, peak alignment, normalization (the ratio of single peak area to total peak area was processed) and deconvolution were performed in Progenesis QI v3.0 to obtain the expression level of each plasma metabolite.

[0047] According to the three chromatographic columns used for separation, the expression levels of the above plasma metabolites were divided into BEH C18 group, HSS T3 group and CSH PhenylHexyl group, that is, each group included: the mass spectrometry data of plasma metabolites separated again in positive ion mode and the mass spectrometry data of plasma metabolites separated again in negative ion mode under the premise of separation by the same chromatographic column. Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed on the expression levels of plasma metabolites in BEH C18 group, HSS T3 group and CSH PhenylHexyl group using SIMCA 14.1 software to obtain variable weight value (VIP) value and OPLS-DA graph. The OPLS-DA graphs of BEH C18 group, HSST3 group and CSH PhenylHexyl group are shown in the figure. Figure 1 As shown by Figure 1 It can be seen that the plasma metabolites of the prostate hyperplasia group and prostate cancer group separated by three chromatographic columns and ion mobility can distinguish patients with prostate hyperplasia and prostate cancer. There are significant differences between the two groups (prostate hyperplasia group and prostate cancer group). Further, through two hundred cross-validations, the determination coefficient (R 2 ) is 0.839, and the prediction ability index (Q 2 ) is -0.539, and the p value is <0.05, which indicates that the results of the OPLS-DA algorithm analysis are not overfitted and the results of the OPLS-DA algorithm analysis are reliable.

[0048] The plasma metabolites separated by the three chromatographic columns were further screened for differential metabolites. The criteria for differential metabolite screening were: variable weight value (VIP) of the first principal component of the OPLS-DA algorithm > 1, T test p value < 0.05, and the absolute value of log2 (FC) greater than 1 (FC represents the difference fold, which is calculated by comparing the expression level of the prostate cancer (PCa) group with that of the prostate hyperplasia group). ACQUITY The screening results of plasma metabolites separated by BEH C18 column are as follows Figure 2 As shown in the "BEHC18" in the figure, ACQUITY The screening results of plasma metabolites separated by HSS T3 column are as follows Figure 2The screening results of plasma metabolites separated by ACQUITY Premier CSH Phenyl-Hexyl Column with VanGuardTM FIT are shown in “HSS T3” in Figure 1. Figure 2 As shown in "CSH Phenyl Hexyl", Figure 2 The rightmost of the two dividing lines represent the differential metabolites that are significantly upregulated in the prostate cancer (PCa) group (p<0.05; log2(FC)>1), the leftmost of the two dividing lines represent the differential metabolites that are significantly downregulated in the prostate hyperplasia group (p<0.05; log2(FC)<-1), and the middle of the two dividing lines represent insignificant differential metabolites. A total of 52 differential metabolites were screened out.

[0049] The mass spectrometry data of differential metabolites were compared with the human metabolome database (HMDB database) using the ChemSpider search engine in Progenesis QI v3.0. The comparison conditions were: the parent ion mass error was less than 5 ppm, the secondary fragment mass error was less than 5 ppm, the isotope similarity was greater than 80%, and the collision cross section (CCS) value deviation was less than 10%. Finally, 52 differential metabolites including PE (20:0 / 15:0) and myristic acid were identified.

[0050] Example 1-4 Data Processing and Analysis

[0051] The ROC curve was drawn by the expression level of PE (20:0 / 15:0), and it was determined that the relative abundance of the best resolution mass spectrum was 1.784, which had a higher diagnostic efficacy. The ROC curve was drawn by the expression level of myristic acid, and it was determined that the relative abundance of the best resolution mass spectrum was 92.54, which had a higher diagnostic efficacy.

[0052] Figure 3 Receiver operating characteristic (ROC) curve for PE (20:0 / 15:0) and TPSA (10 ng / mL) in differentiating patients with prostate cancer and prostate hyperplasia with TPSA>4 ng / mL. Figure 3 As shown, the area under the receiver operating characteristic curve of PE (20:0 / 15:0) for distinguishing between prostate cancer and prostate hyperplasia patients with TPSA>4ng / mL was 0.8618.

[0053] Figure 4 The receiver operating characteristic curves for myristic acid and TPSA (10 ng / mL) for differentiating patients with prostate cancer and prostate hyperplasia with TPSA>4 ng / mL are shown in Table 1. Figure 4As shown, the area under the receiver operating characteristic curve of myristic acid in distinguishing between prostate cancer and prostate hyperplasia patients with TPSA>4ng / mL was 0.8725, which has a large diagnostic value.

[0054] The present invention also performed ROC curve analysis on TPSA (10 ng / mL). The area under the receiver operating characteristic curve (AUC) of TPSA (10 ng / mL) for distinguishing between prostate cancer patients and prostate hyperplasia patients with TPSA>4 ng / ml was 0.6933, indicating that the diagnostic efficacy of PE (20:0 / 15:0) and myristic acid was higher than that of TPSA.

[0055] Plasma PE (20:0 / 15:0) and total prostate-specific antigen (TPSA) were used as PCa diagnostic markers, respectively. The best-resolution mass spectrometry relative abundance of plasma PE (20:0 / 15:0) of 1.784 and total prostate-specific antigen (TPSA) of 10 ng / mL were used as critical values, respectively. 40 participants in the prostate cancer group and 50 participants in the prostate hyperplasia group were screened. The best-resolution mass spectrometry relative abundance of plasma PE (20:0 / 15:0) less than 1.784 was diagnosed as benign prostatic hyperplasia, and greater than or equal to 1.784 was diagnosed as prostate cancer. TPSA less than 10 ng / mL was diagnosed as benign prostatic hyperplasia, and greater than or equal to 10 ng / mL was diagnosed as prostate cancer. The screening results and their sensitivity and specificity were As shown in Table 2, taking the puncture result (N in Table 1) as the gold standard, it can be seen from Table 2 that the sensitivity of plasma PE (20:0 / 15:0) as a PCa diagnostic marker is 85%, and the specificity is 84%. Its sensitivity is equivalent to TPSA (10 ng / mL), but the specificity is greatly improved. The correct index (Youden index) of TPSA (10 ng / mL) is 0.25, which is less than 0.5, and has no diagnostic value. The Youden index of plasma PE (20:0 / 15:0) is 0.69, which is greater than 0.5, and has diagnostic value. This shows that PE (20:0 / 15:0) has a greater diagnostic efficiency than the commonly used TPSA (10 ng / mL) and is a potential biomarker for diagnosing prostate cancer under the premise of TPSA>4 ng / mL.

[0056] Table 2 Sensitivity and specificity of plasma PE (20:0 / 15:0), myristic acid and TPSA (10 ng / mL)

[0057]

[0058]

[0059] Note: Youden index = sensitivity + specificity - 1

[0060] The best resolution mass spectrometry relative abundance of myristic acid of 92.54 and total prostate specific antigen (TPSA) of 10 ng / mL were used as the critical values, respectively, to screen 40 participants in the prostate cancer group and 50 participants in the prostate hyperplasia group. The best resolution mass spectrometry relative abundance of myristic acid less than 92.54 was diagnosed as having benign prostatic hyperplasia, and greater than or equal to 92.54 was diagnosed as having prostate cancer. The TPSA less than 10 ng / mL was diagnosed as having benign prostatic hyperplasia, and greater than or equal to 10 ng / mL was diagnosed as having prostate cancer. The screening results and their sensitivity and specificity are shown in Table 2. As can be seen from Table 2, taking the puncture results (N in Table 1) as the gold standard, the sensitivity of myristic acid as a PCa diagnostic marker is 78%, and the specificity is 92%. Although its sensitivity is slightly lower than TPSA (10 ng / mL), its specificity is greatly improved, and the Youden index of myristic acid is 0.7, which is greater than 0.5, and has diagnostic value. This indicates that myristic acid has a greater diagnostic efficiency than the commonly used TPSA (10 ng / mL) and is a potential biomarker for diagnosing prostate cancer under the premise of TPSA>4 ng / mL.

[0061] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. The use of myristic acid as a marker in the preparation of products for diagnosing prostate cancer.

2. The use according to claim 1, characterized in that: The prerequisite for the use of the product is that the TPSA level in the serum of the patient tested by the product is >4ng / mL.

3. A product for diagnosing prostate cancer, characterized in that: It detects myristic acid in the patient's plasma and uses myristic acid as a marker for diagnosing prostate cancer.

4. The product according to claim 3, characterized in that The prerequisite for the use of the product is that the TPSA level in the serum of the patient tested by the product is >4ng / mL.

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