CN115856157A - Blood or urine metabolic marker and application thereof in endometrial cancer typing - Google Patents
Blood or urine metabolic marker and application thereof in endometrial cancer typing Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a blood or urine metabolic marker and application thereof in endometrial cancer typing. The EC is researched through blood or urine combined metabonomics, the screened combined metabolic marker can distinguish the type I EC from the type II EC, the AUC value can reach 0.938, the sensitivity and the specificity are good, the treatment decision can be participated before an operation, the determination of an operation range is included, the individualized auxiliary treatment is carried out by combining molecular typing after the operation, the relapse is reduced, the survival is prolonged, and the system management of the EC is greatly helped.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a blood or urine metabolic marker and application thereof in endometrial cancer typing.
Background
Traditional typing of Endometrial Cancer (EC) was classified by Bokhman in 1983 into types I and II according to clinical characteristics, epidemiological observations, and hormone-related associations. Type I EC is estrogen dependent, is related to endometrial hyperplasia, is classified into endometrioid carcinoma by world health histology, and is generally high in tissue differentiation and low in invasiveness; type II EC is non-estrogen dependent, and this class is often highly invasive, and histological classification subtypes include serous carcinoma, clear cell carcinoma, mixed adenocarcinoma, and the like. In addition, endometrial cancer also includes some rare subtypes such as mucinous carcinoma, undifferentiated carcinoma, neuroendocrine carcinoma, and the like. Different histological classifications have important guiding significance for determining the operation scheme and postoperative adjuvant therapy. Preoperative curettage or hysteroscopy has the condition of missed diagnosis or misdiagnosis due to the limitation of material taking or sheet making and reading, and the typing differentiation after hysterectomy may be different from that before operation, so that the operation range is insufficient or too large, and difficulty is brought to postoperative adjuvant therapy decision. However, although the postoperative molecular typing is better for individual prognostic guidance, the completed surgical decision cannot be changed, and the molecular typing requires more complicated technology and procedure, is higher in cost, and is difficult to develop in medical units without molecular pathological diagnosis. If EC patients can be distinguished by a minimally invasive or non-invasive method before an operation to carry out layered accurate management and diagnosis and treatment, the system management of EC is very helpful.
Metabonomics has the advantages of sensitive response to physiological and pathological changes of organisms, minimally invasive or non-invasive sampling process, no need of obtaining target organ tissues and the like, and has great potential in the aspect of clinical application. Previous studies found that metabonomics studies can play a role in the staging of EC, and that the concentrations of metabolites in patients in different stages or different pathological types are different, suggesting that certain metabolites can be used to differentiate the severity of the disease. The concentration of some metabolites such as inosine, xanthine and hypoxanthine in a patient with deep muscular layer infiltration is obviously increased, and the metabolites such as hydroxyl sphingomyelin, phosphatidylcholine and endogenous estrogen are related to the muscular layer infiltration depth and can be used as a staging tool of EC. The metabolite biomarkers associated with lymphatic vascular interstitial infiltration included hexadecadienylcarnitine, diacyl residues and C38:1 phosphatidylcholine, diacyl residues and C34:4 phosphatidylcholine and acylalkyl residues and C38:3, and 3-hydroxybutyrate in blood also showed phase-related correlations. However, most of the research samples are small in size and are concentrated in a single blood or urine sample, and the differentiation efficiency of the screened markers is poor.
In view of this, the present invention adopts an Ultra high throughput liquid chromatography-mass spectrometry (UPLC-MS) technique to collect blood and urine samples before EC patients operation for metabonomics combined analysis. Based on the difference between type I EC and type II EC in metabonomics, the invention discusses the pathogenesis of different types of EC by analyzing differential metabolites and metabolic pathways, and the differential metabolites screened by metabonomics can be used as biomarkers for effectively distinguishing type I EC from type II EC.
Disclosure of Invention
Based on the background, the invention provides a blood or urine metabolic marker and application thereof in endometrial cancer typing, aiming at solving the problems that most research samples are small in quantity, mostly concentrated in a single blood or urine sample and the differentiation efficiency of the screened marker is poor.
In order to realize the purpose, the invention is realized by the following technical scheme:
the invention provides application of blood or urine metabolites selected from sucrose, homomannopine D or a combination thereof as diagnostic markers in preparing products for identifying type I and type II endometrial cancer.
Further, the product comprises a kit and a chip.
Further, the product can be used for identifying the type I endometrial cancer and the type II endometrial cancer by detecting the content levels of sucrose and high-mannitol D.
Further, the detection method is a mass spectrometry identification method.
Furthermore, the mass spectrometry adopts a primary full-scan mode for screening and combines secondary targeting analysis. Specifically, the method adopts UPLC-MS to detect the blood or urine metabolites in a full scanning mode, wherein the full scanning mode is to simultaneously acquire primary information of all small molecules in a mass range of 50m/z to 1200m/z, then screen out the differential metabolites through multivariate statistical analysis (principal component analysis PCA and orthogonal partial least squares discriminant analysis OPLS-DA), further perform targeted secondary fragmentation on the differential metabolites, and finally determine the structures of the differential metabolites by combining a database secondary spectrogram.
The invention also provides an identification reagent for identifying the endometrial cancer types I and II, wherein the identification reagent is a reagent for detecting blood or urine metabolites.
Further, the blood or urine metabolite is selected from sucrose, homomannopine D, or a combination thereof.
The invention also provides a kit for identifying type I and type II endometrial cancers, the kit comprising an identification reagent for detecting sucrose, homomannopine D, or a combination thereof.
The invention also provides a chip for identifying type I and type II endometrial cancers, which comprises an identification reagent for detecting sucrose, high-mannitol D or a combination thereof.
Compared with the prior art, the invention has the following beneficial effects:
the EC is researched through blood or urine combined metabonomics, the screened combined metabolic marker can distinguish the type I EC from the type II EC, the AUC value can reach 0.938, the sensitivity and the specificity are good, the treatment decision can be participated before an operation, the determination of an operation range is included, the individualized auxiliary treatment is carried out by combining molecular typing after the operation, the relapse is reduced, the survival is prolonged, and the system management of the EC is greatly helped.
Drawings
FIG. 1 is a taxonomic map of the serum metabolic profiles of type I and type II endometrial cancers (Panel A: PCA taxonomic map, panel B: OPLS-DA taxonomic map);
FIG. 2 is a class diagram of the urine metabolic profile of endometrial cancer types I and II (Panel A: PCA class diagram, panel B: OPLS-DA class diagram);
FIG. 3 is a ROC curve for two metabolite combinations of the present invention identifying type I and type II endometrial cancers.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following more detailed description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Examples
1. Instruments and reagents
1) The instrument comprises: waters ACQUITY H-Class liquid chromatograph (Waters corporation), triple TOF 5600 mass spectrometer (Thermofeisher Scientific corporation).
2) The main reagents are as follows: acetonitrile (Thermofisher Scientific); c18 reverse phase chromatography column (3.0 mm. Times.100 mm, C18, 1.7 μm, waters corporation).
2. Sample collection
1) Blood and urine from 22 patients with type I endometrial cancer and 22 age and gender matched patients with type II endometrial cancer were selected from beijing hospital.
2) Blood collection: the fasting venous blood is extracted, centrifuged for 5min at 3000 g speed, and the supernatant is taken.
3) Collecting urine: collecting fasting morning urine, centrifuging at 5000g for 30min, and removing precipitate.
3. Sample metabolite extraction
1) Blood metabolite extraction
Taking 50 mu L of serum sample, adding 150 mu L of mass spectrum water and 400 mu L of acetonitrile, vortexing, and standing for 1 hour at-20 ℃; centrifuging for 10min at 14000g, centrifuging and concentrating the supernatant, redissolving 50 μ L of 2% acetonitrile water, centrifuging for 10min at 14000g, and sampling 10 μ L after passing through a 10kD filter membrane.
2) Urine metabolite extraction
Collecting 200 μ L urine supernatant, adding 200 μ L acetonitrile, vortexing, standing at 4 deg.C for 30min, centrifuging 14000g for 10min, collecting supernatant, centrifuging, concentrating, redissolving with 200 μ L2% acetonitrile water, centrifuging 14000g for 10min, filtering with 10kD filter membrane, and sampling 10 μ L.
4. Liquid phase analysis
Liquid phase analysis was performed using a Waters acquisition H-Class model ultra performance liquid chromatography system, under the following conditions: and (3) chromatographic column: waters HSS T3C 18 (3.0 mm. Times.100 mm, 1.8 um), column temperature 40 ℃; the mobile phase A is 0.2 per mill formic acid water, the mobile phase B is 0.2 per mill formic acid acetonitrile solution, the elution mode is gradient elution, the elution time is 15 minutes, the flow rate is 0.5 mL/min, and the specific sample injection volume is 10uL. The specific information is shown in table 1 below.
5. Identification by mass spectrometry
Eluted blood or urine metabolites were analyzed using Triple TOF 5600 mass spectrometer with data pattern DDA. The parameters are as follows: the primary full scan range is 50-1200m/z, the integration time is 0.25s, the secondary integration time is 0.1s, GS1 is 55, GS2 is 55, curtain Gas is 35, the temperature is set to 550, and Ionspray Voltage flowing is 4500.
The raw data obtained from the UPLC-LTQ orbitrap was processed using the commercial omics analysis software progenesis QI (Version 2.0, nonlinear Dynamics, UK) from Waters corporation. The software can automatically complete pretreatment procedures such as peak alignment, peak identification, peak correction and the like, and finally output a three-dimensional matrix, namely a spectrum peak index variable consisting of retention time and accurate mass-to-charge ratio, a sample name and peak intensity/area. The obtained data matrix is imported into multivariate statistical software SIMCA-P software 14.0 (Umetrics AB, umea, sweden) for PCA analysis, and the change trend among groups is visualized. Variables with non-reference test p-values less than 0.05 were considered as significantly different variables between groups and were screened as early potential markers of endometrial cancer. And (3) performing secondary fragmentation on the screened differential variables, and selecting 204060eV energy according to specific metabolites by adopting an HCD (High collision fragmentation) fragmentation mode. Deconvoluting the secondary fragment by using progenesis QI software, searching HMDB (HUMAN METABOLOME DATABASE) DATABASE, and determining the structure of the differential metabolite.
6. Screening results
Blood metabolites: unsupervised PCA plots showed that type I and type II endometrial cancers exhibited some discrimination (see fig. 1A); and a model is further constructed by adopting a supervision OPLS-DA, and the two groups are more distinct (see FIG. 1B). 132 blood differential metabolites were screened.
Metabolites of urine: the PCA plot showed a clear distinction between endometrial cancer types I and II (see fig. 2A), and OPLS-DA showed a more clear distinction between the two groups (see fig. 2B). 119 urine differential metabolites are screened out.
The differential metabolites obtained by screening the blood or urine metabolites are subjected to targeted dipolar analysis to determine 2 metabolite structures, namely sucrose and homomannopine D. The method is obtained by analyzing through a logistic regression calculation method, the sucrose and the homomannin D are jointly used for distinguishing the endometrial cancer types I and II, the good prediction effect can be achieved, the AUC value of an ROC curve is 0.938 (see figure 3), 10-fold cross validation AUC is 0.929, and the sensitivity and the specificity are shown in table 2.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (9)
1. Use of a blood or urine metabolite as a diagnostic marker in the manufacture of a product for the identification of type I and type II endometrial cancer, wherein: the blood or urine metabolite is selected from sucrose, homomannopine D, or a combination thereof.
2. Use according to claim 1, characterized in that: the product comprises a kit and a chip.
3. Use according to claim 1, characterized in that: the product can be used for identifying type I and type II endometrial cancer by detecting the content levels of sucrose and high-mannitol D.
4. Use according to claim 3, characterized in that: the detection method is a mass spectrometry identification method.
5. Use according to claim 4, characterized in that: the mass spectrometry identification method adopts a primary full-scanning mode for screening and combines secondary targeting analysis.
6. An identification reagent for identifying type I and type II endometrial cancer, comprising: the identification reagent is a reagent for detecting metabolites in blood or urine.
7. The identification reagent according to claim 6, characterized in that: the blood or urine metabolite is selected from sucrose, homomannopine D, or a combination thereof.
8. A kit for use in the identification of type I and type II endometrial cancer, comprising: the kit comprises the identification reagent according to claim 6 or 7.
9. A chip for identifying type I and type II endometrial cancer, comprising: the chip comprises the identification reagent of claim 6 or 7.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109298115A (en) * | 2018-10-19 | 2019-02-01 | 深圳市绘云生物科技有限公司 | A variety of metabolin quantitative detecting methods and metabolism chip in biological sample |
| CN113156120A (en) * | 2021-03-26 | 2021-07-23 | 中国医学科学院北京协和医院 | Application of B7H4 in preparation of endometrial cancer molecular typing reagent and system |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109298115A (en) * | 2018-10-19 | 2019-02-01 | 深圳市绘云生物科技有限公司 | A variety of metabolin quantitative detecting methods and metabolism chip in biological sample |
| CN113156120A (en) * | 2021-03-26 | 2021-07-23 | 中国医学科学院北京协和医院 | Application of B7H4 in preparation of endometrial cancer molecular typing reagent and system |
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| Title |
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