Application of detection reagents of eight metabolic markers in preparation of breast cancer diagnosis and prognosis products

Abstract

The invention belongs to the technical field of medical examination, and particularly relates to application of detection reagents of eight metabolic markers in preparation of breast cancer diagnosis and prognosis products. The metabolic markers are heptanal, 2-pentanone, 2, 2-dimethyl-propionic acid, 2-decyl decahydronaphthalene, 3, 4, 5, 6, 7, 8-hexahydro-2 (1H)-naphthalenone, 2-[(2-cyano-3, 3-dimethyl cyclopropyl) methyl]-2-methyl-1, 3-dioxolame, methyl isopropyl disulfide and dimethyl silane glycol, and the PubChem CID is 8130, 7895, 6417, 586218, 572673, 560018, 123494 and 11137539 in sequence; research results show that compared with normal healthy people, the combined diagnosis index of the eight metabolic markers in the urine of the breast cancer patient is increased, the eight metabolic markers can be used as the urine marker for predicting the incidence of the breast cancer, the combined evaluation curative effect index of the eight metabolic markers is increased after treatment, and the breast cancer treatment effect can be evaluated. The result provides a specific, rapid and noninvasive detection means for early diagnosis, dynamic monitoring and prognosis of breast cancer, and has wide clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically involving the application of detection reagents for eight metabolic markers in the preparation of products for breast cancer diagnosis and prognosis. Background Technology

[0002] The latest Global Cancer Statistics report (GLOBOCAN) shows that breast cancer (BC) has surpassed lung cancer to become the leading cause of cancer death among women, with approximately 2.3 million new cases in 2020, accounting for 11.7% of all new cancer cases. Furthermore, breast cancer is the fifth leading cause of cancer death in women, accounting for 6.9% of all cancer deaths. Currently, breast cancer screening primarily relies on mammograms and ultrasound examinations. Diagnosis requires a comprehensive differential diagnosis combining clinical manifestations, physical examination, imaging studies, and histopathological examination. Histopathological diagnosis is crucial for confirming and treating breast cancer; however, this technique requires invasive biopsies of breast tissue. Simultaneously, imaging screening, based on morphological changes, often reveals metastatic or advanced stages of cancer, failing to provide early and rapid diagnosis. Therefore, specific, rapid, and non-invasive screening and diagnosis of breast cancer are particularly important for disease prevention and clinical treatment.

[0003] Liquid biopsy offers a non-invasive and minimally invasive method for disease diagnosis and assessment, and it can also monitor disease progression and provide information on treatment efficacy in real time, making it a potential alternative to tissue biopsy. Currently, liquid biopsy of blood samples is widely used to detect solid tumors and immune system diseases, but the potential of urine-based liquid biopsy has not yet been fully explored.

[0004] Metabolomics is a method for measuring all small molecule products of metabolic processes in an organism, and it studies the metabolic profile of unique cellular physiological processes. Urinary metabolomics primarily uses nuclear magnetic resonance (NMR) or mass spectrometry (MS) to broadly identify and analyze small molecules in urine, and screen for highly specific biomarkers. Several studies have shown that differentially expressed metabolites in urine have great potential as biomarkers for early cancer screening and diagnosis.

[0005] In view of this, discovering more highly specific breast cancer biomarkers is the technical problem that this invention aims to solve. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of detection reagents for eight metabolic markers in the preparation of products for breast cancer diagnosis and prognosis.

[0007] The specific solution for implementing the present invention is as follows:

[0008] The application of detection reagents for eight metabolic biomarkers in the preparation of products for breast cancer diagnosis and prognosis, wherein the metabolic biomarkers are heptanal, 2-pentanone, 2,2-dimethylpropionic acid, 2-decyldehydronaphthalene, 3,4,5,6,7,8-hexahydro-2(1H)-naphthone, 2-[(2-cyano-3,3-dimethylcyclopropyl)methyl]-2-methyl-1,3-dioxolane, methyl isopropyl disulfide, and dimethylsilanediol, with PubChem CIDs of 8130, 7895, 6417, 586218, 572673, 560018, 123494, and 11137539, respectively;

[0009] The detection reagent is used to detect the content of the metabolic markers in the sample.

[0010] Furthermore, the sample is the subject's urine.

[0011] Furthermore, the levels of eight metabolic markers in urine were detected using gas chromatography / mass spectrometry.

[0012] Furthermore, the detection reagent includes H2SO4 and an internal standard, wherein the internal standard is a standard of 4-methylpentan-2-ol with a PubChem CID of 7910.

[0013] Furthermore, the H2SO4 and 4-methylpentan-2-ol are present in aqueous solutions, with the concentration of H2SO4 being 5M and the concentration of 4-methylpentan-2-ol being 0.8 mg / mL.

[0014] Furthermore, the breast cancer diagnosis and prognosis product is a kit.

[0015] Furthermore, the kit includes the detection reagent.

[0016] Based on the same inventive concept, the present invention also provides a breast cancer diagnosis and prognosis kit for detecting the aforementioned metabolic biomarkers. The kit includes H2SO4 and an internal standard, wherein the internal standard is a standard of 4-methylpentan-2-ol with a PubChem CID of 7910.

[0017] Furthermore, in the kit, H2SO4 and 4-methylpentan-2-ol are present in aqueous solutions, with the concentration of H2SO4 being 5M and the concentration of 4-methylpentan-2-ol being 0.8 mg / mL.

[0018] The present invention has the following beneficial effects:

[0019] This invention is the first to discover that, compared to healthy controls, breast cancer patients exhibit a significantly elevated combined diagnostic index of eight compounds in their urine: heptanal, 2-pentanone, 2,2-dimethylpropionic acid, 2-decyldehydronaphthyl, 3,4,5,6,7,8-hexahydro-2(1H)-naphthol, 2-[(2-cyano-3,3-dimethylcyclopropyl)methyl]-2-methyl-1,3-dioxolane, methyl isopropyl disulfide, and dimethylsilanediol. This index can distinguish breast cancer from healthy controls, with a specificity of 91.2%, a sensitivity of 100%, an AUC of 0.975, and a cut-off value of -0.529. After treatment of breast cancer patients, a combined efficacy index of eight metabolites was recalculated to evaluate the treatment efficacy. The specificity of this index at one month post-treatment was 97.4%, sensitivity was 87.5%, AUC was 0.963, and cut-off value was 0.355. This index significantly upregulated in breast cancer patients at 1 and 3 months post-treatment, gradually approaching the levels of healthy controls, and can be used as an evaluation indicator for breast cancer treatment efficacy. These results provide a specific, rapid, and non-invasive detection method for early diagnosis, dynamic monitoring, and prognosis of breast cancer, with broad clinical application value. The test reagents in this invention can be mixed with the subject's urine in a single sample, simultaneously and quantitatively detecting multiple metabolic markers with high precision. Sample collection is convenient and painless for patients. The advent of this invention will play a positive role in breast cancer screening and prognosis. Attached Figure Description

[0020] Figure 1 This study examines the changes in a combined diagnostic index of eight metabolites in the urine of healthy controls and breast cancer patients.

[0021] Figure 2 The receiver operating characteristic (ROC) curve is used to represent the combined diagnostic index of eight metabolites as a diagnostic indicator for breast cancer.

[0022] Figure 3 The study assessed changes in efficacy indices by combining eight metabolites in urine of healthy controls and breast cancer patients before treatment and at 1 and 3 months after treatment.

[0023] Figure 4Recipient operating characteristic (ROC) curves were used to evaluate the efficacy of breast cancer treatment at 1 month by using a combined efficacy index of eight metabolites. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0025] Example 1: Screening of Breast Cancer Biomarkers

[0026] 1. Materials and Methods

[0027] 1.1. Research on population and design

[0028] A total of 75 female participants were enrolled, all from the First People's Hospital of Chenzhou City, Hunan Province. Among them, 34 were healthy controls and 41 were breast cancer patients (17 with luminal A, 6 with luminal B, 10 with HER2-positive, and 8 with triple-negative breast cancer). Of these, 5 were stage I, 27 were stage II, and 9 were stage III. There were no statistically significant differences in age, blood pressure, smoking, and medication use between the healthy control group (Control) and the breast cancer group (BC). Specifically, participants were informed and signed informed consent forms before sampling. 10 mL of midstream urine was collected from the first morning urine sample. To minimize the impact of diet on urinary metabolites, participants were required to avoid high-salt, high-fat, and spicy foods the day before sampling and to fast after 8 PM the night before sampling.

[0029] 1.2. Sample Collection and Processing

[0030] After urine sample collection, centrifuge at 5000×g for 30 min to remove precipitate. Take 250 μL of urine, add 50 μL of 5M H2SO4, add 3 μL of 0.8 mg / mL internal standard (4-methylpentan-2-ol, PubChem CID 7910), and mix thoroughly.

[0031] The pretreated sample was placed in an extraction flask, sealed, and then placed on a solid-phase microextraction (SPME) apparatus. The extraction head was a 50 / 30 μm DVB / CAR / PDMS fiber extraction head (Supelco, USA). Equilibration was performed at 25 °C for 10 min. Before extraction, the injection port was aged at 250 °C for 3 min until no impurity peaks were observed. Extraction was performed at 60 °C for 40 min. After extraction, the sample was desorbed at 250 °C for 1 min at the injection port for subsequent analysis.

[0032] 1.3 Non-targeted metabolomics analysis

[0033] Method: Using AB Sciex's TripleTOF TM The 5600+ flight mass spectrometer system was used for electrospray ionization and positive / negative ion modes. Secondary fragmentation was performed using high-energy induced fragmentation (HICF) technology, and unnecessary data was removed using a dynamic exclusion method. Conditions: positive / negative ion spray voltages were 3.4 kV and 2.5 kV, respectively; temperature was 325 °C; scanning mode was full scan; and the scan range was 35–475 m / z.

[0034] After peak identification, filtering, and alignment of the mass spectrometry data using the XCMS program in ProteoWizard (Version 3), information such as mass-to-charge ratio, retention time, and peak area were obtained. The mass-to-charge ratio and retention time were compared against a standard database to obtain the names of specific metabolites. After normalizing the peak area, data dimensionality reduction was performed using PCA. VIP values ​​were calculated using the OPLS-DA model, and differentially expressed metabolites between the healthy control group and the breast cancer group were screened using the Mann-Whitney U test.

[0035] Results: Through database matching, data standardization, quantitative and statistical analysis, it was found that 165 metabolites in the urine of breast cancer patients were significantly different compared with normal healthy people (p<0.05). According to the criteria of VIP>1&FC>1.5&P<0.05, 12 differential metabolites were screened out (Table 1).

[0036] Table 1. Twelve metabolites that showed significant changes in the urine of breast cancer patients.

[0037]

[0038] 1.3 Targeted Gas Chromatography / Mass Spectrometry (GC / MS) Analysis

[0039] The instrument used was a Thermo Fisher Q Exactive GC Orbitrap high-resolution mass spectrometry system (Thermo Fisher, USA), and the chromatographic column was a DB-5ms (60m × 0.25mm × 0.25μm) capillary column (Agilent, USA). The column temperature program was as follows: initial temperature 70℃, hold for 1 min, increase to 100℃ at 12℃ / min, increase to 180℃ at 2℃ / min, hold for 5 min, then increase to 280℃ at 12℃ / min, hold for 2 min. The injection port temperature was 250℃, the carrier gas was helium (He), the flow rate was 1 mL / min, and the split ratio was 1:5.

[0040] The mass spectrometer was an EI ionization source with an ion source temperature of 250℃, a Full MS-SIM scanning mode, a scanning range of 35–475 m / z, and a solvent delay of 0 min.

[0041] 1.4 Result Interpretation

[0042] The mass spectrometer automatically identifies, aligns, and corrects peaks, outputting data including compound name, retention time, precise m / z value, and peak area. Using SPSS (Version 23, IBM, USA) software, the peak areas of eight compounds—heptanal, 2-pentanone, 2,2-dimethylpropionic acid, 2-decyldehydronaphthalene, 3,4,5,6,7,8-hexahydro-2(1H)-naphthone, 2-[(2-cyano-3,3-dimethylcyclopropyl)methyl]-2-methyl-1,3-dioxolane, methyl isopropyl disulfide, and dimethylsilanediol—were calculated using a Lasso regression model. The combined diagnostic index for the eight metabolites was calculated as: 1.750805 + 4.61e-06 * peak area of ​​heptanal - 1.04e-0 The peak area values ​​of 7*2-pentanone were -3.52e-07, 2,2-dimethyl-propionic acid was -1.57e-07, 2-decyldehydronaphthalene was +2.33e-08, 3,4,5,6,7,8-hexahydro-2(1H)-naphthone was +9.16e-08, 2-[(2-cyano-3,3-dimethylcyclopropyl)methyl]-2-methyl-1,3-dioxolane was -2.51e-07, methyl isopropyl disulfide was -2.80e-07, and dimethylsilanediol was also considered. The combined diagnostic index of the eight metabolites was expressed as mean ± standard deviation (Mean ± SD), and a t-test was used to statistically analyze the index in urine from the Control and BC groups. p < 0.05 was considered statistically significant. Then, SPSS software was used to perform Receiver Operating Characteristic (ROC) curve analysis to calculate the specificity, sensitivity, AUC (Areas Under the Curve), and cut-off value of the combined diagnostic index of eight metabolites in indices for breast cancer.

[0043] 2. Results

[0044] Urine mass spectrometry analysis of 34 healthy controls and 41 breast cancer patients yielded combined diagnostic indices of eight metabolites of -2.748±2.802 and 2.695±2.083, respectively. Figure 1As shown, compared with the control group, the combined diagnostic index of eight metabolites in urine was significantly increased in the BC group. The specificity of the combined diagnostic index of eight metabolites in diagnosing breast cancer was 91.2%, the sensitivity was 100%, the AUC value was 0.975, and the cut-off value was -0.529. The ROC curve is shown below. Figure 2 As shown in the figure, this indicates that breast cancer can be diagnosed by detecting eight metabolites in urine using mass spectrometry and calculating the combined diagnostic index of these eight metabolites. This method demonstrates high specificity and sensitivity, and has great potential for broad-spectrum breast cancer diagnosis.

[0045] Therefore, the peak areas of eight metabolites in the urine sample were detected by mass spectrometry. The combined diagnostic index of these eight metabolites was calculated using a formula. If the index is ≥-0.529, the subject is a breast cancer patient; if the index is <-0.529, the subject is not a breast cancer patient. The above diagnostic criteria for breast cancer are for reference only.

[0046] Example 2: Evaluation of the monitoring role of differentially metabolites in breast cancer treatment efficacy

[0047] After obtaining informed consent, urine samples were collected from 33 breast cancer patients one month after treatment and from 22 breast cancer patients three months after treatment. There were no statistically significant differences between the two groups in clinical information such as age, blood pressure, smoking, and medication use. Sample collection, processing, detection, and analysis were performed as in Example 1. The peak areas of eight metabolites detected by mass spectrometry in the urine of 41 breast cancer patients before treatment and 33 breast cancer patients one month after treatment were calculated using a logistic regression model. The combined efficacy evaluation index for the eight metabolites was calculated as: -9.721 - 8.460e-06 * peak area of ​​heptanal - 3.170e-07 * peak area of ​​2-pentanone + 1.013e-05 * peak area of ​​2,2-dimethylpropionic acid + 1.726e- The peak area values ​​are calculated as follows: 06*2-decyldehydronaphthalene + 1.298e-08*3,4,5,6,7,8-hexahydro-2(1H)-naphthone + 2.702e-06*2-[(2-cyano-3,3-dimethylcyclopropyl)methyl]-2-methyl-1,3-dioxolane + 3.967e-09*methyl isopropyl disulfide + 7.093e-07*dimethylsilanediol. Subsequent data analysis and statistical methods are the same as in Example 1.

[0048] Urine mass spectrometry analysis of 34 healthy controls and 41 breast cancer patients before treatment, and 1 and 3 months after treatment in breast cancer patients, yielded efficacy indices for the combined assessment of eight metabolites, which were 6.688±10.410, -5.509±4.773, 2.984±2.611, and 14.600±15.810, respectively. Figure 3As shown, compared with pre-treatment levels, the efficacy index assessed by the combined evaluation of eight metabolites in the urine of breast cancer patients was significantly increased at 1 and 3 months post-treatment. Compared with the healthy control group, the efficacy index assessed by the combined evaluation of eight metabolites in the urine of breast cancer patients was significantly decreased at 1 month post-treatment, but significantly increased at 3 months post-treatment. This indicates that during breast cancer treatment, the efficacy index assessed by the combined evaluation of eight metabolites gradually increases, eventually approaching or even exceeding the level of the healthy control group.

[0049] Meanwhile, the efficacy index for evaluating breast cancer treatment after one month using a combination of eight metabolites showed a specificity of 85.4%, a sensitivity of 93.9%, an AUC value of 0.963, and a cut-off value of 0.355. The ROC curve is shown below. Figure 4 As shown in the figure, the Cut-off value was used to evaluate the efficacy of breast cancer treatment after 3 months, with a specificity of 97.4% and a sensitivity of 87.5%. This indicates that mass spectrometry detection of eight metabolites in urine and calculation of the combined efficacy index of these eight metabolites can be used to assess the efficacy of breast cancer treatment. This method has high specificity and sensitivity, and can dynamically and in real-time monitor the efficacy of breast cancer treatment, thus possessing broad clinical application value.

[0050] Therefore, the peak areas of eight metabolites in the urine sample were detected by mass spectrometry. A combined efficacy evaluation index of these eight metabolites was calculated using a formula. If the index is ≥0.355, the patient is considered to have a good treatment response; if the index is <0.355, the patient is considered to have a poor treatment response. The above efficacy criteria for breast cancer treatment are for reference only.

[0051] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0052] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a reagent for detecting eight metabolic biomarkers in the preparation of products for breast cancer diagnosis and prognosis, characterized in that, The metabolic markers are heptanal, 2-pentanone, 2,2-dimethylpropionic acid, 2-decyldehydronaphthalene, 3,4,5,6,7,8-hexahydro-2(1H)-naphthol, 2-[(2-cyano-3,3-dimethylcyclopropyl)methyl]-2-methyl-1,3-dioxolane, methyl isopropyl disulfide, and dimethylsilanediol, with PubChem CIDs of 8130, 7895, 6417, 586218, 572673, 560018, 123494, and 11137539, respectively. The detection reagent is used to detect the content of the metabolic markers in the sample.

2. The application according to claim 1, characterized in that, The sample was the subject's urine.

3. The application according to claim 2, characterized in that, The levels of eight metabolic markers in urine were detected by gas chromatography / mass spectrometry.

4. The application according to claim 3, characterized in that, The detection reagent includes H2SO4 and an internal standard, wherein the internal standard is a standard of 4-methylpentan-2-ol with a PubChem CID of 7910.

5. The application according to claim 4, characterized in that, The H2SO4 and 4-methylpentan-2-ol were present in aqueous solutions, with the concentration of H2SO4 being 5 M and the concentration of 4-methylpentan-2-ol being 0.8 mg / mL.

6. The application according to claim 5, characterized in that, The breast cancer diagnosis and prognosis product is a kit.

7. The application according to claim 6, characterized in that, The kit includes the detection reagent.

8. A breast cancer diagnosis and prognosis kit, characterized in that, The kit is used to detect the metabolic biomarker of claim 1. The kit includes H2SO4 and an internal standard, wherein the internal standard is a standard of 4-methylpentan-2-ol with a PubChem CID of 7910.

9. A breast cancer diagnosis and prognosis kit according to claim 8, characterized in that, In the kit, H2SO4 and 4-methylpentan-2-ol are present in aqueous solutions, with the concentration of H2SO4 being 5M and the concentration of 4-methylpentan-2-ol being 0.8 mg / mL.

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