WO2023083020A1 - Use of serum metabolic marker for detecting egfr mutation and detection system - Google Patents

Use of serum metabolic marker for detecting egfr mutation and detection system Download PDF

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WO2023083020A1
WO2023083020A1 PCT/CN2022/128169 CN2022128169W WO2023083020A1 WO 2023083020 A1 WO2023083020 A1 WO 2023083020A1 CN 2022128169 W CN2022128169 W CN 2022128169W WO 2023083020 A1 WO2023083020 A1 WO 2023083020A1
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sulfate
acetylcarnitine
dihydrotestosterone
androsterone
epiandrosterone
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PCT/CN2022/128169
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French (fr)
Chinese (zh)
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胡寓旻
姚瑶
黄蓬
关键
张晖
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中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所）
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

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  • the invention relates to the field of medical diagnosis, in particular, it uses serum metabolomics to screen biomarkers to diagnose lung cancer, especially to detect whether the EGFR gene is mutated.
  • Lung cancer is one of the most common malignancies worldwide and one of the cancers with the highest mortality rate. According to the latest data released by my country in 2018, there were 2.1 million new cases of lung cancer in my country, ranking first among malignant tumors, accounting for 18.4% of all tumor deaths (ranking first), and the death toll was 1.8 million (ranking first), accounting for 1.8 million of malignant tumors. cause of death in more than a quarter. It can be seen that lung cancer is a cancer with high morbidity and mortality worldwide. Among them, non-small cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancers. The EGFR gene is one of the most frequently mutated genes in non-small cell lung cancer and is an important driver of the disease. The EGFR mutation rate of Asian lung cancer patients is as high as 40-60%, so EGFR inhibitors are an important targeted therapy for EGFR-mutant lung cancer. The detection of EGFR mutation is particularly important for the choice of treatment.
  • NSCLC non-small cell lung cancer
  • EGFR mutation detection methods mainly rely on invasive methods to extract tumor tissue, such as transbronchial lung biopsy, endotracheal ultrasound-guided transbronchial needle aspiration biopsy, pleural effusion sampling, etc. to obtain lung tissue for gene sequencing. Due to some potential harm caused by invasive surgery and time-consuming, it is clinically necessary to develop a new generation of technology to overcome the limitations of current detection methods.
  • Metabolome refers to all low-molecular-weight metabolites of a certain organism or cell in a specific physiological period. The detection and identification of metabolome can judge the pathophysiological state of the body and find out the markers related to its pathogenesis. Metabolites in serum are stable and quantifiable, which provides a possibility of non-invasive diagnosis for clinical application. Peripheral blood sampling is fast and convenient, which can overcome the limitations of current invasive methods. There is no clinically available serum metabolic marker for the diagnosis of EGFR mutation in lung cancer. This application is of great significance for the development of precise diagnosis of lung cancer and clinical treatment decision-making.
  • the present invention collects serum samples of lung cancer patients with EGFR mutant type (mutation positive, EGFR+) and EGFR wild type (mutation negative, EGFR- or no mutation), and utilizes liquid chromatography-high resolution mass spectrometry (LC-HRMS) to detect The above two types of samples were analyzed by metabolomics and profiling, and two metabolic biomarkers of EGFR+ and EGFR- genotypes in lung cancer were screened out.
  • LC-HRMS liquid chromatography-high resolution mass spectrometry
  • One aspect of the present invention is to provide a method, which screens EGFR+ and EGFR- two different genotypes of lung cancer biomarkers based on serum metabolomics, and the specific steps are as follows:
  • the specific implementation of the step (1) is: the serum samples are from lung cancer serum of two different genotypes of EGFR+ and EGFR- of different sexes and ages.
  • the specific implementation of the step (2) is: the extraction of serum metabolites adopts a three-phase extraction method of methyl tert-butyl ether: methanol: water (10:3:2.5, v/v/v), Add methanol and methyl tert-butyl ether to 50 ⁇ L serum in turn, shake and incubate on ice for 1 hour, then add water, vortex and shake, then centrifuge, take the supernatant and spin dry in a low-temperature vacuum dryer, and the obtained serum metabolism The dried extracts were stored in a -80°C refrigerator.
  • reference serum sample (Reference serum) was batch-processed at the same time as each batch of experimental samples for subsequent signal normalization correction.
  • step (3) reconstitute the dry extract of serum metabolites, take the supernatant after centrifugation to make the sample to be tested, and use liquid chromatography-high resolution mass spectrometry (LC-HRMS) for all samples. ) for detection.
  • LC-HRMS liquid chromatography-high resolution mass spectrometry
  • the m/z ions, retention time and peak area were extracted from the raw data, and the data was normalized. Finally, the database was searched for identification, and the obtained data matrix was used for subsequent analysis.
  • step (4) data filtering is performed on the liquid chromatography-high resolution mass spectrometry data matrix, and the remaining data are grouped by partial least squares discriminant analysis, and two groups of samples can be clearly clustered Group.
  • step (5) is: screening compounds with p-value less than 0.05 and VIP greater than 1 as differential metabolites, and calculating the fold change.
  • step (5) combined with biological significance, the differential metabolic markers of EGFR+ and EGFR- two different genotypes of lung cancer serum were mined, and metabolic pathway analysis was performed.
  • the second aspect of the present invention provides the use of biomarkers in detection reagents for diagnosing lung cancer EGFR gene mutations.
  • the biomarkers are selected from one or more of the following: Indole-3-acrylic acid (3-indole Indole acetic acid); Androsterone sulfate (androsterone sulfate), Dihydrotestosterone sulfate (dihydrotestosterone sulfate), Epiandrosterone sulfate (epiandrosterone sulfate), Etiocholanolone sulfate (this cholanolone sulfate); Ergothioneine (ergothioneine), 3- hydroxydecanoylcarnitine(C10-OH)(3-hydroxydecanoylcarnitine), 3-hydroxyoctanoylcarnitine(C8-OH)(3-hydroxyoctanoylcarnitine), Tetradecenoylcarnitine(C14:
  • the marker consists of the following: Acrylic acid (acrylic acid), Tetradecenoylcarnitine (C14:1) (5-myristoylcarnitine), Indole-3-acrylic acid (3-indole acetic acid), Carnitine (Carnitine), Ergothioneine (Ergothioneine), Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Etiocholanolone Sulfate (Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Cholesterone Sulfate); Acetylcarnitine(C2) (acetylcarnitine).
  • the marker is composed of the following: Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (androsterone sulfate/dihydrotestosterone sulfate/epiandrosterone sulfate/bencholanolone sulfate), Acetylcarnitine ( C2) (acetylcarnitine).
  • the marker consists of the following: Acrylic acid (acrylic acid); 3-Indoleacrylic acid (3-indoleacetic acid); Ergothioneine (ergothioneine); Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Bencholanolone Sulfate); Acetylcarnitine (C2) (Acetylcarnitine).
  • the marker consists of the following: Acrylic acid (acrylic acid); 3-hydroxyoctanoylcarnitine (C8-OH) (3-hydroxyoctanoylcarnitine); 3-Indoleacrylic acid (3-indoleacetic acid), Tetradecenoylcarnitine (C14:1) (5-myristoylcarnitine); Ergothioneine (ergothioneine); Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (androsterone sulfate/dihydrotestosterone sulfate/epiandrosterone sulfate/this sulfate cholanolone); Acetylcarnitine (C2) (acetylcarnitine); Carnitine (carnitine).
  • the marker consists of the following: Indole-3-acrylic acid (3-indole acetic acid); Androsterone sulfate (androsterone sulfate), Dihydrotestosterone sulfate (dihydrotestosterone sulfate), Epiandrosterone sulfate (epiandrosterone sulfate) Androsterone), Etiocholanolone sulfate (the cholanolone sulfate); Ergothioneine (ergothioneine), 3-hydroxydecanoylcarnitine (C10-OH) (3-hydroxydecanoylcarnitine), 3-hydroxyoctanoylcarnitine (C8-OH) (3 -Hydroxyoctanoylcarnitine), Tetradecenoylcarnitine (C14:1) (5-myristoylcarnitine), 3-hydroxybutyrylcarnitine (C4-OH) (-hydroxybutyrylcarnitine (
  • the present invention finds that the increase or down-regulation of some marker substances in serum is related or related to EGFR gene mutation, so that when some marker substances change, the possibility of EGFR gene mutation can be predicted, and then the detection of the gene mutation can be performed. side, so that it can be determined whether the gene is really mutated.
  • mutations in this gene can cause lung cancer, but it does not indicate whether it is associated with other cancers. From another perspective, when it is detected that these marker substances have changed, or have changed significantly, it can be used as the possibility of predicting whether the EGFR gene is mutated. Furthermore, when it is predicted that the possibility of mutation of the EGFR gene increases, the occurrence of diseases associated with the gene, such as the occurrence of lung cancer, can be further predicted.
  • the advantage of the present invention is that: the present invention utilizes the method of serum metabolomics to screen small molecule differential metabolites, which are used as biomarkers for the differential diagnosis of EGFR+ and EGFR- two different genotypes of lung cancer.
  • Another aspect of the present invention provides a system for distinguishing whether the EGFR gene is mutated in patients with lung cancer.
  • the system includes an analysis module, and the analysis module is one or more analysis calculation models listed below:
  • V1- V7 represents in turn: Acrylic acid (acrylic acid), Tetradecenoylcarnitine (C14:1) (5-myristoyl carnitine), Indole-3-acrylic acid (3-indole acetic acid), Carnitine (carnitine), Ergothioneine (ergot Thioneine), Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Etiocholanolone Sulfate (Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Bencholanolone Sulfate); Acetylcarnitine (C2) (acetylcarnitine); or
  • V1-V8 are: Acrylic acid (acrylic acid); 3-hydroxyoctanoylcarnitine (C8-OH) (3-hydroxyoctanoylcarnitine); 3-Indoleacrylic acid (3-indole acetic acid), Tetradecenoylcarnitine (C14:1 )(5-myristoylcarnitine); Ergothioneine (ergothioneine); Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate ; Acetylcarnitine (C2) (acetylcarnitine); Carnitine (carnitine);
  • V1-V10 is: Acrylic acid (acrylic acid), 3-hydroxyoctanoylcarnitine (C8-OH) (3-hydroxyoctanoylcarnitine), 3-hydroxydecanoylcarnitine (C10-OH ) (3-hydroxydecanoylcarnitine); 3-hydroxybutyrylcarnitine (C4-OH) (3-hydroxybutyrylcarnitine); Indole-3-acrylic acid (3-indole acetic acid); Ergothioneine (ergothioneine), Acetylcarnitine (C2) (acetylcarnitine), Androsterone sulfate/Dihydrotestosterone sulf
  • the system also includes an input module, which is to input the content of the marker in the morning.
  • the input is the relative abundance value.
  • FIG. 3 PLS-DA statistical results of lung cancer EGFR mutation positive and EGFR mutation negative (+ESI) and negative spectrum (-ESI).
  • Figure 4 is a graph of the AUC values of model 1.
  • Figure 5 is a graph of the AUC values of model 2.
  • Fig. 6 is a graph of AUC values of model 3.
  • Fig. 7 is a graph of AUC values of model 4.
  • Fig. 8 is a graph of AUC values of model 5.
  • the diagnosis or detection here refers to the detection or assay of the biomarker substances in the sample, or the content of the target biomarker substance, such as absolute content or relative content, and then indicates the presence or quantity of the target marker substance. Whether an individual may have a certain disease, or the possibility of having a certain disease.
  • Diagnosis and detection are used interchangeably here.
  • the result of this test or diagnosis cannot be directly regarded as the direct result of the disease, but an intermediate result. If a direct result is obtained, other auxiliary means such as pathology or anatomy are needed to confirm the disease. disease.
  • the present invention provides a variety of new biomarkers associated with EGFR in lung cancer, and the changes in the content of these markers are directly related to whether EGFR is mutated or the possibility of mutation.
  • connection here means that the appearance or change of a certain biomarker substance in the sample is directly related to a specific disease, such as a relative increase or decrease in the content, which means that the possibility of suffering from this disease is relatively healthy. higher.
  • marker substances are strongly related to disease, some markers are weakly related to disease, or some are not even related to a specific disease.
  • those marker substances with strong correlation it can be used as a marker substance for diagnosing diseases, and those marker substances with weak correlation can be combined with strong marker substances to diagnose a certain disease, increasing the accuracy of test results sex.
  • these markers can be used to distinguish whether EGFR mutation occurs in lung cancer patients or to predict the possibility of mutation.
  • the marker substance here can be directly detected or diagnosed as a single marker substance, and the selection of such a marker substance indicates that the relative change in the content of the marker substance has a strong correlation with lung cancer.
  • one or more marker substances that are strongly associated with lung cancer EGFR can be selected for simultaneous detection.
  • the selection of highly correlated biomarkers for detection or diagnosis can reach a certain standard of accuracy, such as 60%, 65%, 70%, 80%, 85%, 90% or If the accuracy is 95%, it can be explained that these marker substances can obtain the intermediate value for diagnosing a certain disease, but it does not mean that it can directly confirm that you have a certain disease.
  • ROC value can also be selected as a diagnostic marker substance.
  • strong and weak are generally calculated and confirmed by some algorithms, such as the contribution rate or weight analysis of marker substances and lung cancer.
  • Such calculation methods can be significance analysis (p value or FDR value) and fold change (Fold change), multivariate statistical analysis mainly includes principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal Partial least squares discriminant analysis (OPLS-DA), of course, also includes other methods, such as ROC analysis and so on.
  • PCA principal component analysis
  • PLS-DA partial least squares discriminant analysis
  • OPLS-DA orthogonal Partial least squares discriminant analysis
  • the marker substances disclosed in the present invention can be selected, or other existing known marker substances can be selected or combined.
  • Serum samples of lung cancer patients with different genotypes of EGFR+ and EGFR- were collected, including different sexes and age groups. This study collected male and female samples aged 39-78, including EGFR+ (39 cases) and EGFR- (35 cases) lung cancer serum samples of two different genotypes, and matched them according to gender and age.
  • the extraction of serum metabolites adopts the three-phase extraction method of methyl tert-butyl ether: methanol: water (10:3:2.5, v/v/v), and the specific operation is as follows: (1) Place the serum sample on ice to completely melt Finally, take 50uL into a 1.5mL EP tube, add 225 ⁇ L of frozen methanol, and vortex for 30 seconds; (2) add 750 ⁇ L of frozen MTBE, vortex for 30 seconds, shake on ice at 400rpm for 1 hour; (3) add 188 ⁇ L of pure (4) Centrifuge at 15,000 rcf for 10 minutes at 4°C; (5) After centrifugation, take 125 ⁇ L of the supernatant into an EP tube, spin dry with a vacuum freeze dryer, and dry samples of all serum metabolites in Store in -80°C refrigerator before testing.
  • reference serum sample (Reference serum) was batch-processed at the same time as each batch of experimental samples for subsequent data correction.
  • the reference serum samples are selected from 100 cases of healthy people (healthy people refer to normal blood pressure, blood sugar and blood routine, no hepatitis B virus, no obvious diseases in the physical examination results, and no need for medical treatment at present).
  • the 100 cases of healthy people The number of serum males and females is equal, aged 40-55 years old, subjects need to fast overnight and prohibit taking drugs 72 hours before blood collection, excluding those with past disease history and body mass index (BMI) outside the 95th percentile individual.
  • the mixed serum was divided into 50 ⁇ L portions and stored in a -80°C refrigerator.
  • the mass spectrometry instrument model is Q Exactive (Thermo Fisher Scientific Company, USA), using electrospray ionization source (ESI), positive and negative full scan mode (Fullscan) and data-dependent scan mode (ddMS2) for qualitative analysis.
  • the serum sample detection adopts random sampling method. Every 10 serum samples are injected with 1 QC sample test. The first and last needles of the detection sequence are both QC samples. Finally, QC samples were subjected to ddMS2 full scan and segmented scan for compound identification.
  • the raw data of each sample includes total ion current data and mass spectrometry data (as shown in Figure 2), import all sample raw data into Compound Discovery software to obtain m/z ion and retention time information, and search the database (mzCloud and Chemspider) Obtain the compound identification results; further, according to the m/z ion and retention time information, use Tracefinder software to perform chromatographic integration on each sample to obtain more accurate peak area information. Finally, a two-dimensional data matrix containing characteristic ions (combination of m/z ions and retention time) and their content (peak area) is obtained for each sample.
  • Example 4 Using partial least squares discriminant analysis to group samples, combined with significance analysis, to screen differential metabolites of two different genotype groups
  • Metabolomics generally uses a combination of univariate analysis and multivariate statistical analysis to screen differential metabolites, in which univariate analysis mainly includes the significance analysis (p value or FDR value) and fold change (Fold value) of characteristic ions in different groups. change), multivariate statistical analysis mainly includes principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA).
  • PCA principal component analysis
  • PLS-DA partial least squares discriminant analysis
  • OPLS-DA orthogonal partial least squares discriminant analysis
  • variable importance for the projection (Variable Importance for the Projection, VIP) was calculated to measure the influence strength and explanatory power of the expression patterns of each metabolite on the classification and discrimination of samples in each group, and the Wilcoxon rank sum test was further performed to obtain the p value, and according to the group The inner mean was used to calculate the fold change (Fold change, FC) between the two groups.
  • differential metabolites (1) VIP>1; (2) p ⁇ 0.05, that is, when VIP>1 and p ⁇ 0.05, it is determined that there is a significant difference in the metabolite between the two groups, and the metabolite is the difference between the two groups. differential metabolites.
  • Table 1 Differential metabolites grouped by two different genotypes, EGFR+ and EGFR-
  • the P values are all less than 0.05, and some are less than 0.01, indicating that the changes of this gene in lung cancer have significant differences in the metabolic content of some compounds, and some of them reach extremely significant differences.
  • the manifestation of these differences can be used to diagnose the probability of a certain event or predict the probability of occurrence. Therefore, the obvious changes in the above metabolites can at least be used to predict or diagnose the possibility of EGFR gene mutation in lung cancer. This possibility can be 100%, 90%, 80%, 60% and so on. With these prediction results, it can play a positive role in choosing an appropriate treatment plan and prolong the life of the patient.
  • the AUC values of individual metabolites are not the same.
  • the correlation between each metabolite and the mutation of EGFR in lung cancer is not the same.
  • a large AUC value indicates an association cavity, and a small AUC indicates that The correlation is weak.
  • the strength is different, the possibility of these new markers in predicting or diagnosing EGFR gene mutations in lung cancer cannot be denied, and it is also a prediction of health status.
  • a single compound may not be used alone for the diagnosis of the disease, at least it can Explain the health status of the individual, and the probability of EGFR gene mutation in lung cancer patients.
  • Example 4 Model prediction of different marker combinations for diagnosis or distinguishing whether EGFR mutation occurs in patients with lung cancer
  • multiple markers are selected to establish a model prediction.
  • the model selects 7 metabolites for prediction, and the critical value of P is 0.457, that is, the serum detection value (relative abundance) of the above markers is brought into the model 1 equation for calculation.
  • P>0.457 it is identified as EGFR+ genotype ;
  • P ⁇ 0.457 it was identified as EGFR-genotype.
  • Further ROC analysis shown in Figure 4) showed that the AUC of model 1 was 0.801, and the specificity and sensitivity were 0.686 and 0.872, respectively.
  • the critical value of P is 0.548, which is to bring the serum detection value (relative abundance) of the above markers into the model 2 equation for calculation.
  • P>0.548 it is identified as EGFR+ genotype; when P ⁇ 0.548, it is identified as EGFR -genotype.
  • ROC analysis shown in Figure 5 showed that the AUC of model 2 was 0.736, and the specificity and sensitivity were 0.829 and 0.564, respectively.
  • the critical value of P is 0.503, which is to bring the serum detection values (relative abundance) of the above markers into the model 3 equation for calculation.
  • P>0.503 it is identified as EGFR+ genotype; when P ⁇ 0.503, it is identified as EGFR -genotype.
  • ROC analysis shown in Figure 6) showed that the AUC of model 3 was 0.801, and the specificity and sensitivity were 0.714 and 0.872, respectively.
  • the critical value of P is 0.458, which is to bring the serum detection values (relative abundance) of the above markers into the model 4 equation for calculation.
  • P>0.458 it is identified as EGFR+ genotype; when P ⁇ 0.458, it is identified as EGFR -genotype.
  • ROC analysis shown in Figure 7) showed that the AUC of model 4 was 0.807, and the specificity and sensitivity were 0.686 and 0.872, respectively.
  • the critical value of P is 0.409, which is to bring the serum detection values (relative abundance) of the above markers into the model 5 equation for calculation.
  • P>0.409 it is identified as EGFR+ genotype; when P ⁇ 0.409, it is identified as EGFR -genotype.
  • ROC analysis shown in FIG. 8 ) showed that the AUC of model 5 was 0.806, and the specificity and sensitivity were 0.657 and 0.872, respectively.

Abstract

A biomarker is screened out using serum metabolomics, and the screened biomarker is used to diagnose whether there is any mutation in an EGFR gene associated with lung cancer, and used to prepare a system for identifying or diagnosing whether there is any mutation in the EGFR gene associated with lung cancer.

Description

血清代谢标志物作为检测EGFR突变的应用以及检测***Application and detection system of serum metabolic markers as detection of EGFR mutation
本申请要求在先申请中国申请,申请号：202111340149.0，申请日：2021年11月12日的优先权。This application claims the priority of the earlier Chinese application, application number: 202111340149.0, filing date: November 12, 2021.
技术领域technical field
本发明涉及医学诊断领域，具体的说是利用血清代谢组学筛选生物标志物对肺癌进行诊断，尤其是作为检测EGFR基因是否发生突变。The invention relates to the field of medical diagnosis, in particular, it uses serum metabolomics to screen biomarkers to diagnose lung cancer, especially to detect whether the EGFR gene is mutated.
背景技术Background technique
肺癌是全球最常见的恶性肿瘤之一，并且为死亡率最高的癌症之一。2018年我国发布的最新数据显示，我国新发肺癌病例210万，居恶性肿瘤首位，占所有肿瘤死亡病例的18.4％(排名第一)，死亡人数为180万(排名第一)，占恶性肿瘤死因四分之一以上。由此可见肺癌是全球发病率和死亡率双高的癌种。其中非小细胞肺癌(NSCLC)约占所有肺癌的80-85％。EGFR基因是非小细胞肺癌中最常见的突变基因之一，是疾病的重要驱动因素。而亚洲肺癌患者EGFR突变率则高达40-60％，因此EGFR抑制剂是EGFR突变肺癌的重要靶向治疗手段。EGFR突变的检测对于治疗手段的选择显得尤为重要。Lung cancer is one of the most common malignancies worldwide and one of the cancers with the highest mortality rate. According to the latest data released by my country in 2018, there were 2.1 million new cases of lung cancer in my country, ranking first among malignant tumors, accounting for 18.4% of all tumor deaths (ranking first), and the death toll was 1.8 million (ranking first), accounting for 1.8 million of malignant tumors. cause of death in more than a quarter. It can be seen that lung cancer is a cancer with high morbidity and mortality worldwide. Among them, non-small cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancers. The EGFR gene is one of the most frequently mutated genes in non-small cell lung cancer and is an important driver of the disease. The EGFR mutation rate of Asian lung cancer patients is as high as 40-60%, so EGFR inhibitors are an important targeted therapy for EGFR-mutant lung cancer. The detection of EGFR mutation is particularly important for the choice of treatment.
目前，EGFR突变检测手段主要依赖侵入式的方式提取肿瘤组织，如经支气管肺活检、气管内超声引导下经支气管针吸活检、胸水采样等获取肺部组织后进行基因测序。由于侵入性手术带来的一些潜在伤害以及所耗费的时间，临床上需要发展新一代的技术克服目前检测手段的局限。At present, EGFR mutation detection methods mainly rely on invasive methods to extract tumor tissue, such as transbronchial lung biopsy, endotracheal ultrasound-guided transbronchial needle aspiration biopsy, pleural effusion sampling, etc. to obtain lung tissue for gene sequencing. Due to some potential harm caused by invasive surgery and time-consuming, it is clinically necessary to develop a new generation of technology to overcome the limitations of current detection methods.
代谢组(Metabolome)是指某一生物或细胞在特定生理时期内所有的低分子量代谢产物，代谢组的检测与鉴定可以判断机体的病理生理状态，并找出与之发病机制相关的标志物。血清中的代谢物是稳定的并且可以量化的，这为临床应用提供了一种无创诊断的可能性。外周血取样快捷方便，可克服目前侵入式取材的局限性。临床上还没有可用于诊断肺癌EGFR突变的血清代谢标志物，该应用对于发展肺癌精准诊断以及临床治疗决策具有重要意义。Metabolome refers to all low-molecular-weight metabolites of a certain organism or cell in a specific physiological period. The detection and identification of metabolome can judge the pathophysiological state of the body and find out the markers related to its pathogenesis. Metabolites in serum are stable and quantifiable, which provides a possibility of non-invasive diagnosis for clinical application. Peripheral blood sampling is fast and convenient, which can overcome the limitations of current invasive methods. There is no clinically available serum metabolic marker for the diagnosis of EGFR mutation in lung cancer. This application is of great significance for the development of precise diagnosis of lung cancer and clinical treatment decision-making.
发明内容Contents of the invention
本发明通过采集EGFR突变型(突变阳性，EGFR+)和EGFR野生型(突变阴性，EGFR-或者没有突变)的肺癌病人血清样本，利用液相色谱-高分辨质谱联用(LC-HRMS)，对以 上两类样本进行了代谢组学分析与代谢谱图(profiling)的分型，筛选出肺癌中EGFR+和EGFR-两种不同基因型的代谢生物标志物。The present invention collects serum samples of lung cancer patients with EGFR mutant type (mutation positive, EGFR+) and EGFR wild type (mutation negative, EGFR- or no mutation), and utilizes liquid chromatography-high resolution mass spectrometry (LC-HRMS) to detect The above two types of samples were analyzed by metabolomics and profiling, and two metabolic biomarkers of EGFR+ and EGFR- genotypes in lung cancer were screened out.
本发明一方面是提供一种方法，该方法基于血清代谢组学对EGFR+和EGFR-两种不同基因型的肺癌生物标志物进行筛选，具体步骤如下：One aspect of the present invention is to provide a method, which screens EGFR+ and EGFR- two different genotypes of lung cancer biomarkers based on serum metabolomics, and the specific steps are as follows:
(1)收集EGFR+和EGFR-两种不同基因型的肺癌血清样本；(1) Collect EGFR+ and EGFR- lung cancer serum samples of two different genotypes;
(2)提取血清代谢物；(2) extract serum metabolites;
(3)采用液相-质谱联用对提取的血清代谢物进行检测及数据预处理；(3) Detection of extracted serum metabolites and data preprocessing by liquid chromatography-mass spectrometry;
(4)利用偏最小二乘判别分析对样本分群，结合显著性分析，筛选两组间的差异代谢物或者差异的生物标志物；(4) Use partial least squares discriminant analysis to group samples, combined with significance analysis, to screen differential metabolites or differential biomarkers between the two groups;
(5)根据筛选得到的差异代谢物，挖掘肺癌的生物标志物以及这些标志物的应用。(5) Based on the screened differential metabolites, discover the biomarkers of lung cancer and the application of these markers.
在一些方式中，所述步骤(1)的具体实现为：血清样本来自不同性别和年龄的EGFR+和EGFR-两种不同基因型的肺癌血清。In some manners, the specific implementation of the step (1) is: the serum samples are from lung cancer serum of two different genotypes of EGFR+ and EGFR- of different sexes and ages.
在一些方式中，所述步骤(2)的具体实现为：血清代谢物的提取采用甲基叔丁基醚:甲醇:水(10:3:2.5，v/v/v)三相萃取方法，向50μL血清中依次加入甲醇、甲基叔丁基醚，冰上震荡孵化1小时后，再加入水，涡旋震荡后离心，取下层清液到低温真空干燥仪中旋干，得到的血清代谢物干提物放置-80℃冰箱中保存。In some ways, the specific implementation of the step (2) is: the extraction of serum metabolites adopts a three-phase extraction method of methyl tert-butyl ether: methanol: water (10:3:2.5, v/v/v), Add methanol and methyl tert-butyl ether to 50 μL serum in turn, shake and incubate on ice for 1 hour, then add water, vortex and shake, then centrifuge, take the supernatant and spin dry in a low-temperature vacuum dryer, and the obtained serum metabolism The dried extracts were stored in a -80°C refrigerator.
本研究在每批实验样本处理的同时都随批处理一个参比血清样本(Reference serum)，以用于后续信号归一化校正。In this study, a reference serum sample (Reference serum) was batch-processed at the same time as each batch of experimental samples for subsequent signal normalization correction.
所述步骤(3)的具体实现为：对血清代谢物干提物进行复溶，离心后取上清液制成待测样本，所有样本采用液相色谱-高分辨质谱联用(LC-HRMS)进行检测。对原始数据提取m/z离子、保留时间和峰面积，并进行数据归一化，最后搜索数据库鉴定，得到的数据矩阵进行后续分析。The specific implementation of the step (3) is: reconstitute the dry extract of serum metabolites, take the supernatant after centrifugation to make the sample to be tested, and use liquid chromatography-high resolution mass spectrometry (LC-HRMS) for all samples. ) for detection. The m/z ions, retention time and peak area were extracted from the raw data, and the data was normalized. Finally, the database was searched for identification, and the obtained data matrix was used for subsequent analysis.
进一步地，所述步骤(4)的具体实现为：对液相色谱-高分辨质谱数据矩阵进行数据过滤，剩余数据利用偏最小二乘判别分析对样本分群，两组样本可得到明显的聚类分群。Further, the specific implementation of the step (4) is: data filtering is performed on the liquid chromatography-high resolution mass spectrometry data matrix, and the remaining data are grouped by partial least squares discriminant analysis, and two groups of samples can be clearly clustered Group.
在一些方式中，所述步骤(5)的具体实现为：筛选p值小于0.05同时VIP大于1的化合物作为差异代谢物，并计算倍数变化。另外，结合生物学意义，挖掘EGFR+和EGFR-两种不同基因型的肺癌血清的差异代谢标志物，并进行代谢通路分析。In some ways, the specific implementation of step (5) is: screening compounds with p-value less than 0.05 and VIP greater than 1 as differential metabolites, and calculating the fold change. In addition, combined with biological significance, the differential metabolic markers of EGFR+ and EGFR- two different genotypes of lung cancer serum were mined, and metabolic pathway analysis was performed.
本发明的第二方面，提供生物标志物在用于诊断肺癌EGFR基因是否突变检测试剂中的用途，所述生物标志物选择如下的一种或者几种：Indole-3-acrylic acid(3-吲哚乙酸)； Androsterone sulfate(硫酸雄酮)，Dihydrotestosterone sulfate(硫酸二氢睾酮)，Epiandrosterone sulfate(硫酸表雄酮)，Etiocholanolone sulfate(硫酸本胆烷醇酮)；Ergothioneine(麦角硫因)，3-hydroxydecanoylcarnitine(C10-OH)(3-羟基癸酰肉碱)，3-hydroxyoctanoylcarnitine(C8-OH)(3-羟基辛酰肉碱)，Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)，3-hydroxybutyrylcarnitine(C4-OH)(-羟基丁酰肉碱)，Acetylcarnitine(C2)(乙酰肉碱)，Carnitine(肉碱)，Acrylic acid(丙烯酸)。The second aspect of the present invention provides the use of biomarkers in detection reagents for diagnosing lung cancer EGFR gene mutations. The biomarkers are selected from one or more of the following: Indole-3-acrylic acid (3-indole Indole acetic acid); Androsterone sulfate (androsterone sulfate), Dihydrotestosterone sulfate (dihydrotestosterone sulfate), Epiandrosterone sulfate (epiandrosterone sulfate), Etiocholanolone sulfate (this cholanolone sulfate); Ergothioneine (ergothioneine), 3- hydroxydecanoylcarnitine(C10-OH)(3-hydroxydecanoylcarnitine), 3-hydroxyoctanoylcarnitine(C8-OH)(3-hydroxyoctanoylcarnitine), Tetradecenoylcarnitine(C14:1)(5-myristoylcarnitine), 3 -Hydroxybutyrylcarnitine (C4-OH) (-hydroxybutyrylcarnitine), Acetylcarnitine (C2) (acetylcarnitine), Carnitine (carnitine), Acrylic acid (acrylic acid).
在一些方式中，所述的标记物由下列组成：Acrylic acid(丙烯酸)，Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)，Indole-3-acrylic acid(3-吲哚乙酸)，Carnitine(肉碱)，Ergothioneine(麦角硫因)，Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)；Acetylcarnitine(C2)(乙酰肉碱)。In some forms, the marker consists of the following: Acrylic acid (acrylic acid), Tetradecenoylcarnitine (C14:1) (5-myristoylcarnitine), Indole-3-acrylic acid (3-indole acetic acid), Carnitine (Carnitine), Ergothioneine (Ergothioneine), Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Etiocholanolone Sulfate (Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Cholesterone Sulfate); Acetylcarnitine(C2) (acetylcarnitine).
在一些方式中，所述的标记物由下列组成：Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)，Acetylcarnitine(C2)(乙酰肉碱)。In some ways, the marker is composed of the following: Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (androsterone sulfate/dihydrotestosterone sulfate/epiandrosterone sulfate/bencholanolone sulfate), Acetylcarnitine ( C2) (acetylcarnitine).
在一些方式中，所述的标记物由下列组成：Acrylic acid(丙烯酸)；3-Indoleacrylic acid(3-吲哚乙酸)；Ergothioneine(麦角硫因)；Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)；Acetylcarnitine(C2)(乙酰肉碱)。In some embodiments, the marker consists of the following: Acrylic acid (acrylic acid); 3-Indoleacrylic acid (3-indoleacetic acid); Ergothioneine (ergothioneine); Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Bencholanolone Sulfate); Acetylcarnitine (C2) (Acetylcarnitine).
在一些方式中，所述的标记物由下列组成：Acrylic acid(丙烯酸)；3-hydroxyoctanoylcarnitine(C8-OH)(3-羟基辛酰肉碱)；3-Indoleacrylic acid(3-吲哚乙酸)，Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)；Ergothioneine(麦角硫因)；Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)；Acetylcarnitine(C2)(乙酰肉碱)；Carnitine(肉碱)。In some forms, the marker consists of the following: Acrylic acid (acrylic acid); 3-hydroxyoctanoylcarnitine (C8-OH) (3-hydroxyoctanoylcarnitine); 3-Indoleacrylic acid (3-indoleacetic acid), Tetradecenoylcarnitine (C14:1) (5-myristoylcarnitine); Ergothioneine (ergothioneine); Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (androsterone sulfate/dihydrotestosterone sulfate/epiandrosterone sulfate/this sulfate cholanolone); Acetylcarnitine (C2) (acetylcarnitine); Carnitine (carnitine).
在一些方式中，所述的标记物由下列组成：Indole-3-acrylic acid(3-吲哚乙酸)；Androsterone sulfate(硫酸雄酮)，Dihydrotestosterone sulfate(硫酸二氢睾酮)，Epiandrosterone sulfate(硫酸表雄酮)，Etiocholanolone sulfate(硫酸本胆烷醇酮)；Ergothioneine(麦角硫因)，3-hydroxydecanoylcarnitine(C10-OH)(3-羟基癸酰肉碱)，3-hydroxyoctanoylcarnitine(C8-OH)(3-羟基辛酰肉碱)，Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)， 3-hydroxybutyrylcarnitine(C4-OH)(-羟基丁酰肉碱)，Acetylcarnitine(C2)(乙酰肉碱)，Carnitine(肉碱)，Acrylic acid(丙烯酸)。也就是说，本发明发现一些血清中的标记物质的含量增加或者下调与EGFR基因突变有关系或者关联，这样当一些标记物质变化，可以预测EGFR基因突变的可能性，然后进行该基因突变的检侧，从而可以确定是否是该基因真的发生了突变。In some forms, the marker consists of the following: Indole-3-acrylic acid (3-indole acetic acid); Androsterone sulfate (androsterone sulfate), Dihydrotestosterone sulfate (dihydrotestosterone sulfate), Epiandrosterone sulfate (epiandrosterone sulfate) Androsterone), Etiocholanolone sulfate (the cholanolone sulfate); Ergothioneine (ergothioneine), 3-hydroxydecanoylcarnitine (C10-OH) (3-hydroxydecanoylcarnitine), 3-hydroxyoctanoylcarnitine (C8-OH) (3 -Hydroxyoctanoylcarnitine), Tetradecenoylcarnitine (C14:1) (5-myristoylcarnitine), 3-hydroxybutyrylcarnitine (C4-OH) (-hydroxybutyrylcarnitine), Acetylcarnitine (C2) (acetylcarnitine), Carnitine (carnitine), Acrylic acid (acrylic acid). That is to say, the present invention finds that the increase or down-regulation of some marker substances in serum is related or related to EGFR gene mutation, so that when some marker substances change, the possibility of EGFR gene mutation can be predicted, and then the detection of the gene mutation can be performed. side, so that it can be determined whether the gene is really mutated.
可以理解，该基因突变可以引起肺癌，但并不表示与别的癌症是否关联。从另一个方明，当检测到这些标记物质发生了变化后，或者显著变化后，可以作为预测EGFR基因是否发生突变的可能性。进一步，当预测EGFR基因发生突变的可能性增加，则可以进一步预测与该基因关联的疾病的发生，例如肺癌的发生。It can be understood that mutations in this gene can cause lung cancer, but it does not indicate whether it is associated with other cancers. From another perspective, when it is detected that these marker substances have changed, or have changed significantly, it can be used as the possibility of predicting whether the EGFR gene is mutated. Furthermore, when it is predicted that the possibility of mutation of the EGFR gene increases, the occurrence of diseases associated with the gene, such as the occurrence of lung cancer, can be further predicted.
本发明的优势在于：本发明利用血清代谢组学的方法筛选出小分子差异代谢物，作为生物标志物，用于EGFR+和EGFR-两种不同基因型的肺癌的鉴别诊断。The advantage of the present invention is that: the present invention utilizes the method of serum metabolomics to screen small molecule differential metabolites, which are used as biomarkers for the differential diagnosis of EGFR+ and EGFR- two different genotypes of lung cancer.
本发明的另一方面，提供一种区分肺癌患者EGFR基因是否突变的***，所述的***包括分析模块，所述的分析模块为下一列一种或者几种分析计算模型：Another aspect of the present invention provides a system for distinguishing whether the EGFR gene is mutated in patients with lung cancer. The system includes an analysis module, and the analysis module is one or more analysis calculation models listed below:
模型1方程为：ln[P/(1-P)]＝0.64×V1-0.34×V2-0.37×V3-0.44×V4-0.68×V5-0.89×V6-1.32×V7+3.23；其中，V1-V7分别顺次表示：Acrylic acid(丙烯酸)，Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)，Indole-3-acrylic acid(3-吲哚乙酸)，Carnitine(肉碱)，Ergothioneine(麦角硫因)，Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)；Acetylcarnitine(C2)(乙酰肉碱)；或者The equation of model 1 is: ln[P/(1-P)]＝0.64×V1-0.34×V2-0.37×V3-0.44×V4-0.68×V5-0.89×V6-1.32×V7+3.23; among them, V1- V7 represents in turn: Acrylic acid (acrylic acid), Tetradecenoylcarnitine (C14:1) (5-myristoyl carnitine), Indole-3-acrylic acid (3-indole acetic acid), Carnitine (carnitine), Ergothioneine (ergot Thioneine), Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Etiocholanolone Sulfate (Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Bencholanolone Sulfate); Acetylcarnitine (C2) (acetylcarnitine); or
模型2方程为：ln[P/(1-P)]＝-0.06×V1-0.68×V2+0.83；其中，V1-V2顺次为：Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)，Acetylcarnitine(C2)(乙酰肉碱)； Model 2 equation is: ln[P/(1-P)]=-0.06×V1-0.68×V2+0.83; wherein, V1-V2 is: Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (androsterone sulfate Ketone/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Cholesterone Sulfate), Acetylcarnitine (C2) (acetylcarnitine);
模型3方程为：ln[P/(1-P)]＝0.08×V1-0.18×V2-0.29×V3-0.49×V4-0.1.25×V5+2.08；其中V1-V5顺次为：Acrylic acid(丙烯酸)；3-Indoleacrylic acid(3-吲哚乙酸)；Ergothioneine(麦角硫因)；Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)；Acetylcarnitine(C2)(乙酰肉碱)；The equation of model 3 is: ln[P/(1-P)]＝0.08×V1-0.18×V2-0.29×V3-0.49×V4-0.1.25×V5+2.08; where V1-V5 is in sequence: Acrylic acid (acrylic acid); 3-Indoleacrylic acid (3-indole acetic acid); Ergothioneine (ergothioneine); Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (androsterone sulfate/dihydrotestosterone sulfate/epiandrosterone sulfate/sulfate This cholanolone); Acetylcarnitine (C2) (acetylcarnitine);
模型4方程为：ln[P/(1-P)]＝1.06×V1+0.65×V2-0.51×V3-1.06×V4-1.11×V5-1.26×V6-1.26×V7-1.42×V8+4.8；其中，V1-V8顺次为：Acrylic acid(丙烯酸)； 3-hydroxyoctanoylcarnitine(C8-OH)(3-羟基辛酰肉碱)；3-Indoleacrylic acid(3-吲哚乙酸)，Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)；Ergothioneine(麦角硫因)；Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)；Acetylcarnitine(C2)(乙酰肉碱)；Carnitine(肉碱)；Model 4 equation is: ln[P/(1-P)]=1.06×V1+0.65×V2-0.51×V3-1.06×V4-1.11×V5-1.26×V6-1.26×V7-1.42×V8+4.8; Among them, V1-V8 are: Acrylic acid (acrylic acid); 3-hydroxyoctanoylcarnitine (C8-OH) (3-hydroxyoctanoylcarnitine); 3-Indoleacrylic acid (3-indole acetic acid), Tetradecenoylcarnitine (C14:1 )(5-myristoylcarnitine); Ergothioneine (ergothioneine); Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate ; Acetylcarnitine (C2) (acetylcarnitine); Carnitine (carnitine);
或者，模型5方程为：ln[P/(1-P)]＝1.17×V1+0.84×V2+0.29×V3+0.06×V4-0.56×V5-1.26×V6-1.39×V7-1.4×V8-1.4×V9-1.69×V10+5.31，其中，V1-V10顺次为：Acrylic acid(丙烯酸)，3-hydroxyoctanoylcarnitine(C8-OH)(3-羟基辛酰肉碱)，3-hydroxydecanoylcarnitine(C10-OH)(3-羟基癸酰肉碱)；3-hydroxybutyrylcarnitine(C4-OH)(3-羟基丁酰肉碱)；Indole-3-acrylic acid(3-吲哚乙酸)；Ergothioneine(麦角硫因)，Acetylcarnitine(C2)(乙酰肉碱)，Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)；Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)，Carnitine(肉碱)。Alternatively, the model 5 equation is: ln[P/(1-P)]=1.17×V1+0.84×V2+0.29×V3+0.06×V4-0.56×V5-1.26×V6-1.39×V7-1.4×V8- 1.4×V9-1.69×V10+5.31, among them, V1-V10 is: Acrylic acid (acrylic acid), 3-hydroxyoctanoylcarnitine (C8-OH) (3-hydroxyoctanoylcarnitine), 3-hydroxydecanoylcarnitine (C10-OH ) (3-hydroxydecanoylcarnitine); 3-hydroxybutyrylcarnitine (C4-OH) (3-hydroxybutyrylcarnitine); Indole-3-acrylic acid (3-indole acetic acid); Ergothioneine (ergothioneine), Acetylcarnitine (C2) (acetylcarnitine), Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (androsterone sulfate/dihydrotestosterone sulfate/epiandrosterone sulfate/benzcholanolone sulfate); Tetradecenoylcarnitine(C14:1) (5-myristoylcarnitine), Carnitine (carnitine).
在一些方式中，该***还包括输入模块，就是输入上午标志物的含量。当采用液相色谱-高分辨质谱联用检测以上标志物的时候，输入的是相对丰度值。In some aspects, the system also includes an input module, which is to input the content of the marker in the morning. When using liquid chromatography-high resolution mass spectrometry to detect the above markers, the input is the relative abundance value.
附图说明Description of drawings
图1分析流程图Figure 1 Analysis flow chart
图2总离子流图和质谱图Figure 2 Total ion chromatogram and mass spectrum
图3肺癌EGFR突变阳性与EGFR突变阴性的(+ESI)与负谱(-ESI)的PLS-DA统计结果图。Fig. 3 PLS-DA statistical results of lung cancer EGFR mutation positive and EGFR mutation negative (+ESI) and negative spectrum (-ESI).
图4是模型1的AUC值图。Figure 4 is a graph of the AUC values of model 1.
图5是模型2的AUC值图。Figure 5 is a graph of the AUC values of model 2.
图6是模型3的AUC值图。Fig. 6 is a graph of AUC values of model 3.
图7是模型4的AUC值图。Fig. 7 is a graph of AUC values of model 4.
图8是模型5的AUC值图。Fig. 8 is a graph of AUC values of model 5.
详细说明Detailed description
(1)诊断或者检测(1) Diagnosis or detection
这里的诊断或者检测是指对于样本中的生物标记物质进行检测或者化验，或者目的生物标记物质的含量，例如绝对含量或者相对含量，然后通过目标标记物质是否存在或者数 量的多少来说明提供样本的个体是否可能具有患某种疾病，或者具有某种疾病的可能性。The diagnosis or detection here refers to the detection or assay of the biomarker substances in the sample, or the content of the target biomarker substance, such as absolute content or relative content, and then indicates the presence or quantity of the target marker substance. Whether an individual may have a certain disease, or the possibility of having a certain disease.
这里的诊断与检测的含义可以互换。这种检测的结果或者诊断的结果是不能直接作为患病的直接结果，而是一种中间结果，如果获得直接的结果，还需通过病理学或者解剖学等其它辅助手段才能确认患有某种疾病。例如，本发明提供了多种与肺癌EGFR具有关联性的新的生物标记物质，这些标记物质的含量的变化与是否EGFR发生突变或者突变的可能性直接的关联性。Diagnosis and detection are used interchangeably here. The result of this test or diagnosis cannot be directly regarded as the direct result of the disease, but an intermediate result. If a direct result is obtained, other auxiliary means such as pathology or anatomy are needed to confirm the disease. disease. For example, the present invention provides a variety of new biomarkers associated with EGFR in lung cancer, and the changes in the content of these markers are directly related to whether EGFR is mutated or the possibility of mutation.
(2)标记物质与肺癌EGFR突变的联系(2) The relationship between marker substances and EGFR mutations in lung cancer
这里的联系是指某种生物标记物质在样本中出现或者含量的变化与特定疾病具有直接的关联性，例如含量的相对升高或者降低，表示这种患有这种疾病的可能性相对健康人员更高。The connection here means that the appearance or change of a certain biomarker substance in the sample is directly related to a specific disease, such as a relative increase or decrease in the content, which means that the possibility of suffering from this disease is relatively healthy. higher.
如果样本中多个不同的标记物质同时出现或者含量的相对变化，表示这种患有这种疾病的可能性相对健康人员也更高。也就是说标记物质种类中，某一些标记物质与患病的关联性强，有些标记物与患病的关联性弱，或者有些甚至与某种特定的疾病无关联。对于那些关联性强的标记物质中的一种或者多种，可以作为诊断疾病的标记物质，与那些关联性弱的标记物质可以与强的标记物质组合来诊断某种疾病，增加检测结果的准确性。If multiple different marker substances appear in the sample at the same time or the relative content changes, it means that the possibility of suffering from this disease is higher than that of healthy people. That is to say, among the types of marker substances, some marker substances are strongly related to disease, some markers are weakly related to disease, or some are not even related to a specific disease. For one or more of those marker substances with strong correlation, it can be used as a marker substance for diagnosing diseases, and those marker substances with weak correlation can be combined with strong marker substances to diagnose a certain disease, increasing the accuracy of test results sex.
针对本发明发现的血清中的众多生物标记物质，这些标记物质都可以用来进行区分肺癌患EGFR是否发生突变或者预测突变的可能性。这里的标记物质可以单独作为单个的标记物质来进行直接的检测或者诊断，选择这样的标记物质表示该标记物质的含量的相对变化与肺癌具有强的关联性。当然，可以理解的是，可以选择与肺癌EGFR关联性强的一种或者多种标记物物质的同时检测。正常的理解是，在一些方式中，选择关联性强的生物标记物质来进行检测或者诊断可以达到一定标准的准确定，例如60％，65％，70％，80％，85％，90％或者95％的准确性，则可以说明，这些标记物质可以获得诊断某中疾病的中间值，但并不表示就能直接确认患有某中疾病。For the numerous biomarkers in the serum discovered in the present invention, these markers can be used to distinguish whether EGFR mutation occurs in lung cancer patients or to predict the possibility of mutation. The marker substance here can be directly detected or diagnosed as a single marker substance, and the selection of such a marker substance indicates that the relative change in the content of the marker substance has a strong correlation with lung cancer. Of course, it can be understood that one or more marker substances that are strongly associated with lung cancer EGFR can be selected for simultaneous detection. The normal understanding is that in some ways, the selection of highly correlated biomarkers for detection or diagnosis can reach a certain standard of accuracy, such as 60%, 65%, 70%, 80%, 85%, 90% or If the accuracy is 95%, it can be explained that these marker substances can obtain the intermediate value for diagnosing a certain disease, but it does not mean that it can directly confirm that you have a certain disease.
当然，也可以选择ROC值越大的作为诊断的标志物质。所谓的强，弱一般通过一些算法来计算确认，例如标记物物质与肺癌的贡献率或者权重分析。这样的计算方法可以是显著性分析(p值或FDR值)和倍数变化(Fold change)，多元统计分析主要包括主成分分析(PCA)、偏最小二乘判别分析(PLS-DA)和正交偏最小二乘判别分析(OPLS-DA)，当然还包括其他的方法，例如ROC分析等等。当然，其它的模型预测方法也是可以的，在具体选择生物标志物的时候，可以选择本发明所公开的标志物质，也可以选择或者结合 其它现有公知的标志物质。Of course, one with a larger ROC value can also be selected as a diagnostic marker substance. The so-called strong and weak are generally calculated and confirmed by some algorithms, such as the contribution rate or weight analysis of marker substances and lung cancer. Such calculation methods can be significance analysis (p value or FDR value) and fold change (Fold change), multivariate statistical analysis mainly includes principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal Partial least squares discriminant analysis (OPLS-DA), of course, also includes other methods, such as ROC analysis and so on. Of course, other model prediction methods are also possible. When specifically selecting biomarkers, the marker substances disclosed in the present invention can be selected, or other existing known marker substances can be selected or combined.
具体实施方式Detailed ways
为了更为具体地描述本发明，下面结合附图及具体实施方式对本发明的技术方案进行详细说明。这些说明仅仅是表明本发明是如何实现的，并不能限定本发明的具体范围。本发明的范围在权利要求中限定。In order to describe the present invention more specifically, the technical solutions of the present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. These descriptions only show how the present invention is realized, and cannot limit the specific scope of the present invention. The scope of the present invention is defined in the claims.
实施例1：收集血清样本Example 1: Collection of Serum Samples
收集了EGFR+和EGFR-两种不同基因型的肺癌病人血清样本，包括不同性别和年龄段。本研究收集了年龄在39-78岁之间的男性和女性样本，包括EGFR+(39例)和EGFR-(35例)两种不同基因型的肺癌血清样本，按照性别与年龄段匹配。Serum samples of lung cancer patients with different genotypes of EGFR+ and EGFR- were collected, including different sexes and age groups. This study collected male and female samples aged 39-78, including EGFR+ (39 cases) and EGFR- (35 cases) lung cancer serum samples of two different genotypes, and matched them according to gender and age.
实施例2：提取血清代谢物Example 2: Extraction of serum metabolites
血清代谢物的提取采用甲基叔丁基醚:甲醇:水(10:3:2.5,v/v/v)三相萃取方法，具体操作如下：(1)将血清样本置于冰上完全融解后，取50uL到1.5mL EP管中，加入225μL冷冻甲醇，涡旋30秒；(2)再加入750μL冷冻MTBE，涡旋30秒后，400rpm冰上震荡1小时；(3)再加入188μL纯水，涡旋1分钟；(4)4℃下以15000rcf离心10分钟；(5)离心后，取125μL下层清液到EP管中，用真空冷冻干燥仪旋干，所有血清代谢物干样在测试前放置-80℃冰箱存储。The extraction of serum metabolites adopts the three-phase extraction method of methyl tert-butyl ether: methanol: water (10:3:2.5, v/v/v), and the specific operation is as follows: (1) Place the serum sample on ice to completely melt Finally, take 50uL into a 1.5mL EP tube, add 225μL of frozen methanol, and vortex for 30 seconds; (2) add 750μL of frozen MTBE, vortex for 30 seconds, shake on ice at 400rpm for 1 hour; (3) add 188μL of pure (4) Centrifuge at 15,000 rcf for 10 minutes at 4°C; (5) After centrifugation, take 125 μL of the supernatant into an EP tube, spin dry with a vacuum freeze dryer, and dry samples of all serum metabolites in Store in -80°C refrigerator before testing.
本研究在每批实验样本处理的同时都随批处理一个参比血清样本(Reference serum)，以用于后续数据校正。参比血清样本是选取了100例健康人(健康人指血压血糖血常规均正常且无乙肝病毒，体检结果无提示明显疾病，目前无需就诊治疗的人群)血清混合而成，这100例健康人血清的男性与女性数量相等，年龄40-55岁，受检者需禁食一夜并在采血前72小时禁止服用药物，排除有过往疾病史及体重指数(BMI)在第95百分位之外的个体。混合后的血清以每份50μL分装，置于-80℃冰箱保存。In this study, a reference serum sample (Reference serum) was batch-processed at the same time as each batch of experimental samples for subsequent data correction. The reference serum samples are selected from 100 cases of healthy people (healthy people refer to normal blood pressure, blood sugar and blood routine, no hepatitis B virus, no obvious diseases in the physical examination results, and no need for medical treatment at present). The 100 cases of healthy people The number of serum males and females is equal, aged 40-55 years old, subjects need to fast overnight and prohibit taking drugs 72 hours before blood collection, excluding those with past disease history and body mass index (BMI) outside the 95th percentile individual. The mixed serum was divided into 50 μL portions and stored in a -80°C refrigerator.
实施例3：对提取的血清代谢物进行检测及数据预处理Example 3: Detection of extracted serum metabolites and data preprocessing
(1)血清代谢物的复溶：向血清干提物中加入120μL复溶溶剂(乙腈:水＝4:1)，涡旋震荡5分钟后，4℃下以15000×g离心10分钟，取100μL上清液于内衬管制成待测样本。(1) Reconstitution of serum metabolites: Add 120 μL of reconstitution solvent (acetonitrile:water=4:1) to the dry serum extract, vortex and shake for 5 minutes, centrifuge at 15000×g for 10 minutes at 4°C, and take 100 μL of supernatant was used to make the sample to be tested in the liner tube.
(2)QC样本：所有肺癌、良性肺结节和健康人血清待测样本各取10μL，涡旋震荡混合均匀后，制成QC样本。(2) QC samples: Take 10 μL each of the serum samples to be tested from lung cancer, benign pulmonary nodules, and healthy people, vortex and oscillate to mix evenly, and make QC samples.
(3)样本检测方法：用液相色谱-高分辨质谱联用(LC-HRMS)进行检测。(3) Sample detection method: detection by liquid chromatography-high resolution mass spectrometry (LC-HRMS).
①液相色谱条件① Liquid chromatography conditions
色谱柱：BEH Amide(100×2.1mm,1.7μm)。Chromatographic column: BEH Amide (100×2.1mm, 1.7μm).
流动相：正模式下，A相为乙腈:水＝95:5(10mM醋酸铵,0.1％甲酸)，B相为乙腈:水＝50:50(10mM醋酸铵,0.1％甲酸)；负模式下，A相为乙腈:水＝95:5(10mM醋酸铵,pH＝9.0,氨水调节)，B相为乙腈:水＝50:50(10mM醋酸铵,pH＝9.0,氨水调节)。Mobile phase: In positive mode, phase A is acetonitrile:water=95:5 (10mM ammonium acetate, 0.1% formic acid), phase B is acetonitrile:water=50:50 (10mM ammonium acetate, 0.1% formic acid); in negative mode , Phase A is acetonitrile:water=95:5 (10mM ammonium acetate, pH=9.0, adjusted with ammonia water), phase B is acetonitrile:water=50:50 (10mM ammonium acetate, pH=9.0, adjusted with ammonia water).
洗脱梯度如下表1所示：The elution gradient is shown in Table 1 below:
表1：LC-HRMS流动相洗脱梯度Table 1: LC-HRMS mobile phase elution gradient
Figure PCTCN2022128169-appb-000001
Figure PCTCN2022128169-appb-000001
②质谱条件② Mass Spectrometry Conditions
质谱仪器型号为Q Exactive(美国Thermo Fisher Scientific公司)，采用电喷雾离子源(ESI)、正负全扫描模式(Fullscan)和数据依赖性扫描模式(ddMS2)进行定性分析。喷雾电压+3800/-3200V；雾化温度350℃；高纯氮气作为鞘气和辅气，参数分别设置为40arb和10arb；离子传输管温度320℃；质量扫描范围70-1050m/z；一级扫描分辨率为70000FWHM，二级扫描分辨率为35000FWHM。The mass spectrometry instrument model is Q Exactive (Thermo Fisher Scientific Company, USA), using electrospray ionization source (ESI), positive and negative full scan mode (Fullscan) and data-dependent scan mode (ddMS2) for qualitative analysis. Spray voltage +3800/-3200V; atomization temperature 350°C; high-purity nitrogen as sheath gas and auxiliary gas, the parameters are set to 40arb and 10arb respectively; ion transfer tube temperature 320°C; mass scanning range 70-1050m/z; The scanning resolution is 70000FWHM, and the secondary scanning resolution is 35000FWHM.
③进样方法③Injection method
每次检测前先进6针QC样本以稳定检测***，血清样本检测采取随机进样的方式，每进样10针血清样本***1针QC样本测试，检测序列的第一针和最后一针均为QC样本。最后对QC样本进行ddMS2全扫描和分段扫描以用于化合物鉴定。Six QC samples are advanced before each test to stabilize the detection system. The serum sample detection adopts random sampling method. Every 10 serum samples are injected with 1 QC sample test. The first and last needles of the detection sequence are both QC samples. Finally, QC samples were subjected to ddMS2 full scan and segmented scan for compound identification.
(4)数据预处理(4) Data preprocessing
①原始数据矩阵①Original data matrix
每个样本的原始数据都包括总离子流数据和质谱数据(如图2所示)，将所有样本原始数据导入Compound Discovery软件得到m/z离子和保留时间信息，并搜索数据库(mzCloud和Chemspider)得到化合物鉴定结果；进一步地根据m/z离子和保留时间信息，使用 Tracefinder软件对各个样本进行色谱积分，得到更准确的峰面积信息。最终每个样本都得到一个包含特征离子(m/z离子和保留时间的组合)及其含量(峰面积)的二维数据矩阵。The raw data of each sample includes total ion current data and mass spectrometry data (as shown in Figure 2), import all sample raw data into Compound Discovery software to obtain m/z ion and retention time information, and search the database (mzCloud and Chemspider) Obtain the compound identification results; further, according to the m/z ion and retention time information, use Tracefinder software to perform chromatographic integration on each sample to obtain more accurate peak area information. Finally, a two-dimensional data matrix containing characteristic ions (combination of m/z ions and retention time) and their content (peak area) is obtained for each sample.
②数据缺失值的剔除和插补② Elimination and imputation of data missing values
代谢组学原始数据矩阵中经常有数据缺失值，这主要与检测背景噪音、质谱峰提取和峰对齐方法等有关，太多的零或缺失值会给下游分析带来困难，因此一般将所有样本中缺失值大于50％的特征离子剔除，其余化合物的缺失值进行插补，本研究使用MetaboAnalyst5.0分析软件进行缺失值处理，选择K-Nearest Neighbours(KNN)的方式插补。There are often data missing values in the original data matrix of metabolomics, which is mainly related to the detection of background noise, mass spectrum peak extraction and peak alignment methods, etc. Too many zero or missing values will bring difficulties to downstream analysis, so generally all samples The characteristic ions with missing values greater than 50% were eliminated, and the missing values of the remaining compounds were imputed. In this study, MetaboAnalyst5.0 analysis software was used for missing value processing, and K-Nearest Neighbors (KNN) was selected for imputation.
③数据校正和过滤③ Data correction and filtering
本研究利用随批处理的一个参比血清作为一个天然的“类内标”，将各前处理批次的实验样本原始数据基于对应批次的参比血清的数据进行归一化信号校正，得到各特征离子的相对丰度，并删除在QC样本中RSD>30％的特征离子，得到的最终的分析数据矩阵。In this study, a reference serum that was processed with the batch was used as a natural "class internal standard", and the original data of the experimental samples of each pre-treatment batch were corrected based on the data of the corresponding batch of reference serum to obtain The relative abundance of each characteristic ion, and delete the characteristic ion with RSD>30% in the QC sample, to obtain the final analysis data matrix.
实施例4：利用偏最小二乘判别分析对样本分群，结合显著性分析，筛选两种不同基因型分组的差异代谢物Example 4: Using partial least squares discriminant analysis to group samples, combined with significance analysis, to screen differential metabolites of two different genotype groups
代谢组学一般采用单变量分析和多元统计分析结合的方式进行差异代谢物的筛选，其中单变量分析主要包括特征离子在不同分组中的显著性分析(p值或FDR值)和倍数变化(Fold change)，多元统计分析主要包括主成分分析(PCA)、偏最小二乘判别分析(PLS-DA)和正交偏最小二乘判别分析(OPLS-DA)等。Metabolomics generally uses a combination of univariate analysis and multivariate statistical analysis to screen differential metabolites, in which univariate analysis mainly includes the significance analysis (p value or FDR value) and fold change (Fold value) of characteristic ions in different groups. change), multivariate statistical analysis mainly includes principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA).
在进行统计分析之前，需对数据进行适当的数据归一化、转换和缩放。本研究使用MetaboAnalyst 5.0分析软件进行统计分析，并进行数据归一化(Normalization by the sum)、转换(Log transformation)和缩放(Auto scaling)。对EGFR+和EGFR-两种不同基因型分组进行偏最小二乘判别分析(PLS-DA)(如图3所示)，得到明显的分群结果。同时，计算变量投影重要度(Variable Importance for the Projection，VIP)以衡量各代谢物的表达模式对各组样本分类判别的影响强度和解释能力，进一步进行Wilcoxon秩和检验得到p值，并根据组内平均值计算两组间的倍数变化(Fold change，FC)。Appropriate data normalization, transformation, and scaling of the data is required prior to statistical analysis. This study uses MetaboAnalyst 5.0 analysis software for statistical analysis, and data normalization (Normalization by the sum), transformation (Log transformation) and scaling (Auto scaling). Partial least squares discriminant analysis (PLS-DA) was performed on EGFR+ and EGFR- two different genotype groups (as shown in Figure 3), and obvious grouping results were obtained. At the same time, the variable importance for the projection (Variable Importance for the Projection, VIP) was calculated to measure the influence strength and explanatory power of the expression patterns of each metabolite on the classification and discrimination of samples in each group, and the Wilcoxon rank sum test was further performed to obtain the p value, and according to the group The inner mean was used to calculate the fold change (Fold change, FC) between the two groups.
根据差异代谢物的筛选标准：(1)VIP>1；(2)p<0.05，即VIP>1且p<0.05时，判定代谢物在两组间存在显著差异，该代谢物为两组间的差异代谢物。According to the screening criteria for differential metabolites: (1) VIP>1; (2) p<0.05, that is, when VIP>1 and p<0.05, it is determined that there is a significant difference in the metabolite between the two groups, and the metabolite is the difference between the two groups. differential metabolites.
本发明发现主要显著的差异代谢物有下表1所示。The main significant differential metabolites found in the present invention are shown in Table 1 below.
表1:EGFR+和EGFR-两种不同基因型分组的差异代谢物Table 1: Differential metabolites grouped by two different genotypes, EGFR+ and EGFR-
Figure PCTCN2022128169-appb-000002
Figure PCTCN2022128169-appb-000002
Figure PCTCN2022128169-appb-000003
Figure PCTCN2022128169-appb-000003
从以上分析可以看出，P值都小于0.05，有些是小于0.01，说明肺癌中该基因的变化，对于某些化合物的代谢含量具有显著差异，有些达到极显著差异的。这些差异的体现就可以用来进行诊断某种事件发生的概率或者预测发生的概率的高低。所以，以上代谢物发生了明显的变化，至少可以用来预测或者诊断肺癌EGFR基因发生突变的可能性，这种可能性可以是100％，90％，80％，60％等。有了这些预测结果，可以对选择合适的治疗方案起到积极的作用，延长患者的生命。From the above analysis, it can be seen that the P values are all less than 0.05, and some are less than 0.01, indicating that the changes of this gene in lung cancer have significant differences in the metabolic content of some compounds, and some of them reach extremely significant differences. The manifestation of these differences can be used to diagnose the probability of a certain event or predict the probability of occurrence. Therefore, the obvious changes in the above metabolites can at least be used to predict or diagnose the possibility of EGFR gene mutation in lung cancer. This possibility can be 100%, 90%, 80%, 60% and so on. With these prediction results, it can play a positive role in choosing an appropriate treatment plan and prolong the life of the patient.
表：2EGFR+和EGFR-两种不同基因型差异代谢物的单个ROC分析结果Table: Single ROC analysis results of differential metabolites of 2EGFR+ and EGFR- two different genotypes
Figure PCTCN2022128169-appb-000004
Figure PCTCN2022128169-appb-000004
Figure PCTCN2022128169-appb-000005
Figure PCTCN2022128169-appb-000005
从以上结果看，单个代谢物的AUC值并不相同，表面每一个代谢物质与肺癌EGFR发生突变与否的关联性强弱不一样，AUC值大的，表示关联性腔，AUC小的，表示关联性弱。虽然强弱不一样，但是不能否定这些新的标志物在预测或者诊断肺癌EGFR基因突变的可能性，也是一种健康状况的预测，虽然单个化合物不一定单独用来对疾病的诊断，但是至少可以说明个体的健康状况，表面肺癌患者的EGFR基因突变的概率问题。From the above results, the AUC values of individual metabolites are not the same. On the surface, the correlation between each metabolite and the mutation of EGFR in lung cancer is not the same. A large AUC value indicates an association cavity, and a small AUC indicates that The correlation is weak. Although the strength is different, the possibility of these new markers in predicting or diagnosing EGFR gene mutations in lung cancer cannot be denied, and it is also a prediction of health status. Although a single compound may not be used alone for the diagnosis of the disease, at least it can Explain the health status of the individual, and the probability of EGFR gene mutation in lung cancer patients.
实施例4：不同标志物组合进行诊断或者区分肺癌患者EGFR是否发生突变的模型预测Example 4: Model prediction of different marker combinations for diagnosis or distinguishing whether EGFR mutation occurs in patients with lung cancer
为了更加准确的对肺癌EGFR是否发生突变进行鉴别或者诊断,选取多个标志物建立模型预测.In order to more accurately identify or diagnose whether EGFR mutation occurs in lung cancer, multiple markers are selected to establish a model prediction.
模型1:Model 1:
Figure PCTCN2022128169-appb-000006
Figure PCTCN2022128169-appb-000006
模型1方程为：ln[P/(1-P)]＝0.64×V1-0.34×V2-0.37×V3-0.44×V4-0.68×V5-0.89×V6-1.32×V7+3.23。该模型选择7中代谢物进行预测,P的临界值为0.457，即将上述标志物的血清检测值(相对丰度)带入模型1方程中进行计算，当P>0.457时，鉴别为EGFR+基因型；当P≤0.457时，鉴别为EGFR-基因型。进一步进行ROC分析(图4所示)，模型1的AUC为0.801，特异性与敏感性分别为0.686、0.872。The equation of model 1 is: ln[P/(1-P)]=0.64×V1-0.34×V2-0.37×V3-0.44×V4-0.68×V5-0.89×V6-1.32×V7+3.23. The model selects 7 metabolites for prediction, and the critical value of P is 0.457, that is, the serum detection value (relative abundance) of the above markers is brought into the model 1 equation for calculation. When P>0.457, it is identified as EGFR+ genotype ; When P≤0.457, it was identified as EGFR-genotype. Further ROC analysis (shown in Figure 4) showed that the AUC of model 1 was 0.801, and the specificity and sensitivity were 0.686 and 0.872, respectively.
模型2:Model 2:
Figure PCTCN2022128169-appb-000007
Figure PCTCN2022128169-appb-000007
Figure PCTCN2022128169-appb-000008
Figure PCTCN2022128169-appb-000008
模型2方程为：ln[P/(1-P)]＝-0.06×V1-0.68×V2+0.83。The model 2 equation is: ln[P/(1-P)]=-0.06×V1-0.68×V2+0.83.
P的临界值为0.548，即将上述标志物的血清检测值(相对丰度)带入模型2方程中进行计算，当P>0.548时，鉴别为EGFR+基因型；当P≤0.548时，鉴别为EGFR-基因型。进一步进行ROC分析(图5所示)，模型2的AUC为0.736，特异性与敏感性分别为0.829、0.564。The critical value of P is 0.548, which is to bring the serum detection value (relative abundance) of the above markers into the model 2 equation for calculation. When P>0.548, it is identified as EGFR+ genotype; when P≤0.548, it is identified as EGFR -genotype. Further ROC analysis (shown in Figure 5) showed that the AUC of model 2 was 0.736, and the specificity and sensitivity were 0.829 and 0.564, respectively.
模型3:Model 3:
Figure PCTCN2022128169-appb-000009
Figure PCTCN2022128169-appb-000009
模型3方程为：ln[P/(1-P)]＝0.08×V1-0.18×V2-0.29×V3-0.49×V4-0.1.25×V5+2.08。The equation of model 3 is: ln[P/(1-P)]=0.08×V1-0.18×V2-0.29×V3-0.49×V4-0.1.25×V5+2.08.
P的临界值为0.503，即将上述标志物的血清检测值(相对丰度)带入模型3方程中进行计算，当P>0.503时，鉴别为EGFR+基因型；当P≤0.503时，鉴别为EGFR-基因型。进一步进行ROC分析(图6所示)，模型3的AUC为0.801，特异性与敏感性分别为0.714、0.872。The critical value of P is 0.503, which is to bring the serum detection values (relative abundance) of the above markers into the model 3 equation for calculation. When P>0.503, it is identified as EGFR+ genotype; when P≤0.503, it is identified as EGFR -genotype. Further ROC analysis (shown in Figure 6) showed that the AUC of model 3 was 0.801, and the specificity and sensitivity were 0.714 and 0.872, respectively.
模型4model 4
Figure PCTCN2022128169-appb-000010
Figure PCTCN2022128169-appb-000010
Figure PCTCN2022128169-appb-000011
Figure PCTCN2022128169-appb-000011
模型4方程为：ln[P/(1-P)]＝1.06×V1+0.65×V2-0.51×V3-1.06×V4-1.11×V5-1.26×V6-1.26×V7-1.42×V8+4.8。The equation of model 4 is: ln[P/(1-P)]=1.06×V1+0.65×V2-0.51×V3-1.06×V4-1.11×V5-1.26×V6-1.26×V7-1.42×V8+4.8.
P的临界值为0.458，即将上述标志物的血清检测值(相对丰度)带入模型4方程中进行计算，当P>0.458时，鉴别为EGFR+基因型；当P≤0.458时，鉴别为EGFR-基因型。进一步进行ROC分析(图7所示)，模型4的AUC为0.807，特异性与敏感性分别为0.686、0.872。The critical value of P is 0.458, which is to bring the serum detection values (relative abundance) of the above markers into the model 4 equation for calculation. When P>0.458, it is identified as EGFR+ genotype; when P≤0.458, it is identified as EGFR -genotype. Further ROC analysis (shown in Figure 7) showed that the AUC of model 4 was 0.807, and the specificity and sensitivity were 0.686 and 0.872, respectively.
模型5 model 5
Figure PCTCN2022128169-appb-000012
Figure PCTCN2022128169-appb-000012
Figure PCTCN2022128169-appb-000013
Figure PCTCN2022128169-appb-000013
模型5方程为：ln[P/(1-P)]＝1.17×V1+0.84×V2+0.29×V3+0.06×V4-0.56×V5-1.26×V6-1.39×V7-1.4×V8-1.4×V9-1.69×V10+5.31。The equation of model 5 is: ln[P/(1-P)]＝1.17×V1+0.84×V2+0.29×V3+0.06×V4-0.56×V5-1.26×V6-1.39×V7-1.4×V8-1.4× V9-1.69×V10+5.31.
P的临界值为0.409，即将上述标志物的血清检测值(相对丰度)带入模型5方程中进行计算，当P>0.409时，鉴别为EGFR+基因型；当P≤0.409时，鉴别为EGFR-基因型。进一步进行ROC分析(图8所示)，模型5的AUC为0.806，特异性与敏感性分别为0.657、0.872。The critical value of P is 0.409, which is to bring the serum detection values (relative abundance) of the above markers into the model 5 equation for calculation. When P>0.409, it is identified as EGFR+ genotype; when P≤0.409, it is identified as EGFR -genotype. Further ROC analysis (shown in FIG. 8 ) showed that the AUC of model 5 was 0.806, and the specificity and sensitivity were 0.657 and 0.872, respectively.
本发明说明书中提到的所有专利和出版物都表示这些是本领域的公开技术，本发明可以使用。这里所引用的所有专利和出版物都被同样列在参考文献中，跟每一个出版物具体的单独被参考引用一样。这里所述的本发明可以在缺乏任何一种元素或多种元素，一种限制或多种限制的情况下实现，这里这种限制没有特别说明。例如这里每一个实例中术语“包含”，“实质由……组成”和“由……组成”可以用两者之一的其余2个术语代替。这里的所谓的“一个”仅仅表示“一”的意思，而不排除仅仅只是包括一个，也可以表示包括2个以上。这里采用的术语和表达方式所为描述方式，而不受其限制，这里也没有任何意图来指明此书描述的这些术语和解释排除了任何等同的特征，但是可以知道，可以在本发明和权利要求的范围内做任何合适的改变或修改。可以理解，本发明所描述的实施例子都是 一些优选的实施例子和特点，任何本领域的一般技术人员都可以根据本发明描述的精髓下做一些更改和变化，这些更改和变化也被认为属于本发明的范围和独立权利要求以及附属权利要求所限制的范围内。All patents and publications mentioned in the specification of the present invention indicate that these are disclosed technologies in the art, and the present invention can be used. All patents and publications cited herein are likewise listed in the references as if each publication were specifically and individually referenced. The invention described herein can be practiced in the absence of any element or elements, limitation or limitations, no such limitation being specifically stated herein. For example, the terms "comprising", "consisting essentially of" and "consisting of" in each instance herein can be replaced by one of the other two terms. The so-called "one" here only means "one", and it does not exclude that only one is included, and it may also mean that more than two are included. The terms and expressions used here are descriptive, not limiting, and there is no intention to indicate that these terms and explanations described in this book exclude any equivalent features, but it can be known that they can be used in the present invention and rights make any appropriate changes or modifications within the scope of the requirements. It can be understood that the implementation examples described in the present invention are some preferred implementation examples and characteristics, and any person skilled in the art can make some modifications and changes according to the spirit described in the present invention, and these modifications and changes are also considered to belong to The scope of the invention is as defined by the independent and appended claims.

Claims (9)

  1. 生物标志物在用于诊断肺癌EGFR基因是否突变检测试剂中的用途，所述生物标志物选择如下的一种或者几种：Indole-3-acrylic acid(3-吲哚乙酸)，Androsterone sulfate(硫酸雄酮)，Dihydrotestosterone sulfate(硫酸二氢睾酮)，Epiandrosterone sulfate(硫酸表雄酮)，Etiocholanolone sulfate(硫酸本胆烷醇酮)，Ergothioneine(麦角硫因)，3-hydroxydecanoylcarnitine(C10-OH)(3-羟基癸酰肉碱)，3-hydroxyoctanoylcarnitine(C8-OH)(3-羟基辛酰肉碱)，Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)，3-hydroxybutyrylcarnitine(C4-OH)(-羟基丁酰肉碱)，Acetylcarnitine(C2)(乙酰肉碱)，Carnitine(肉碱)，Acrylic acid(丙烯酸)。The use of biomarkers in the detection reagent for diagnosing lung cancer EGFR gene mutation, the biomarkers are selected from one or more of the following: Indole-3-acrylic acid (3-indole acetic acid), Androsterone sulfate (sulfuric acid Androsterone), Dihydrotestosterone sulfate (dihydrotestosterone sulfate), Epiandrosterone sulfate (epiandrosterone sulfate), Etiocholanolone sulfate (cholanolone sulfate), Ergothioneine (ergothioneine), 3-hydroxydecanoylcarnitine (C10-OH) (3 -Hydroxydecanoylcarnitine), 3-hydroxyoctanoylcarnitine (C8-OH) (3-hydroxyoctanoylcarnitine), Tetradecenoylcarnitine (C14:1) (5-myristoylcarnitine), 3-hydroxybutyrylcarnitine (C4-OH) ( - Hydroxybutyrylcarnitine), Acetylcarnitine (C2) (acetylcarnitine), Carnitine (carnitine), Acrylic acid (acrylic acid).
  2. 根据权利要求1所述的用途，所述的标记物由下列组成：Acrylic acid(丙烯酸)，Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)，Indole-3-acrylic acid(3-吲哚乙酸)，Carnitine(肉碱)，Ergothioneine(麦角硫因)，Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮),Acetylcarnitine(C2)(乙酰肉碱)。The use according to claim 1, the marker is made up of the following: Acrylic acid (acrylic acid), Tetradecenoylcarnitine (C14:1) (5-myristoylcarnitine), Indole-3-acrylic acid (3-indole Acetic acid), Carnitine (carnitine), Ergothioneine (ergothioneine), Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (androsterone sulfate/dihydrotestosterone sulfate/epiandrosterone sulfate/bencholanolone sulfate), Acetylcarnitine (C2) (acetylcarnitine).
  3. 根据权利要求1所述的用途，所述的标记物由下列组成：Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)，Acetylcarnitine(C2)(乙酰肉碱)。Use according to claim 1, the marker is made up of the following: Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (androsterone sulfate/dihydrotestosterone sulfate/epiandrosterone sulfate/benzcholanolone sulfate ), Acetylcarnitine (C2) (acetylcarnitine).
  4. 根据权利要求1所述的用途，所述的标记物由下列组成：Acrylic acid(丙烯酸),3-Indoleacrylic acid(3-吲哚乙酸)，Ergothioneine(麦角硫因),Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)，Acetylcarnitine(C2)(乙酰肉碱)。purposes according to claim 1, described marker is made up of following: Acrylic acid (acrylic acid), 3-Indoleacrylic acid (3-indole acetic acid), Ergothioneine (ergothioneine), Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone Sulfate/Etiocholanolone sulfate (Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Bencholanolone Sulfate), Acetylcarnitine (C2) (acetylcarnitine).
  5. 根据权利要求1所述的用途，所述的标记物由下列组成：Acrylic acid(丙烯酸)，3-hydroxyoctanoylcarnitine(C8-OH)(3-羟基辛酰肉碱)，3-Indoleacrylic acid(3-吲哚乙酸)，Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)，Ergothioneine(麦角硫因)，Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)，Acetylcarnitine(C2)(乙酰肉碱)，Carnitine(肉碱)。The use according to claim 1, the marker is made up of the following: Acrylic acid (acrylic acid), 3-hydroxyoctanoylcarnitine (C8-OH) (3-hydroxyoctanoylcarnitine), 3-Indoleacrylic acid (3-indoleacrylic acid) Indole acetic acid), Tetradecenoylcarnitine (C14:1) (5-myristoylcarnitine), Ergothioneine (ergothioneine), Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (androsterone sulfate/dihydrotestosterone sulfate/epandrosterone sulfate Ketone/Sulphate), Acetylcarnitine (C2) (acetylcarnitine), Carnitine (carnitine).
  6. 根据权利要求1所述的用途，所述的标记物由下列组成：Indole-3-acrylic acid(3-吲哚乙酸)，Androsterone sulfate(硫酸雄酮)，Dihydrotestosterone sulfate(硫酸二氢睾酮)，Epiandrosterone sulfate(硫酸表雄酮)，Etiocholanolone sulfate(硫酸 本胆烷醇酮)，Ergothioneine(麦角硫因)，3-hydroxydecanoylcarnitine(C10-OH)(3-羟基癸酰肉碱)，3-hydroxyoctanoylcarnitine(C8-OH)(3-羟基辛酰肉碱)，Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)，3-hydroxybutyrylcarnitine(C4-OH)(-羟基丁酰肉碱)，Acetylcarnitine(C2)(乙酰肉碱)，Carnitine(肉碱)，Acrylicacid(丙烯酸)。purposes according to claim 1, described marker is made up of following: Indole-3-acrylic acid (3-indole acetic acid), Androsterone sulfate (androsterone sulfate), Dihydrotestosterone sulfate (dihydrotestosterone sulfate), Epiandrosterone sulfate (epiandrosterone sulfate), Etiocholanolone sulfate (cholanolone sulfate), Ergothioneine (ergothioneine), 3-hydroxydecanoylcarnitine (C10-OH) (3-hydroxydecanoylcarnitine), 3-hydroxyoctanoylcarnitine (C8- OH)(3-hydroxyoctanoylcarnitine), Tetradecenoylcarnitine(C14:1)(5-myristoylcarnitine), 3-hydroxybutyrylcarnitine(C4-OH)(-hydroxybutyrylcarnitine), Acetylcarnitine(C2)(acetyl Carnitine), Carnitine (carnitine), Acrylic acid (acrylic acid).
  7. 一种区分肺癌患者EGFR基因是否突变的***，所述的***包括分析模块，所述的分析模块为下一列一种或者几种分析计算模型：A system for distinguishing whether the EGFR gene is mutated in patients with lung cancer, the system includes an analysis module, and the analysis module is one or several analysis calculation models listed below:
    模型1方程为：ln[P/(1-P)]＝0.64×V1-0.34×V2-0.37×V3-0.44×V4-0.68×V5-0.89×V6-1.32×V7+3.23；其中，V1-V7分别顺次表示：Acrylic acid(丙烯酸)，Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)，Indole-3-acrylic acid(3-吲哚乙酸)，Carnitine(肉碱)，Ergothioneine(麦角硫因)，Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)，Acetylcarnitine(C2)(乙酰肉碱)；The equation of model 1 is: ln[P/(1-P)]＝0.64×V1-0.34×V2-0.37×V3-0.44×V4-0.68×V5-0.89×V6-1.32×V7+3.23; among them, V1- V7 represents in turn: Acrylic acid (acrylic acid), Tetradecenoylcarnitine (C14:1) (5-myristoyl carnitine), Indole-3-acrylic acid (3-indole acetic acid), Carnitine (carnitine), Ergothioneine (ergot Thioneine), Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Etiocholanolone Sulfate (Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Bencholanolone Sulfate), Acetylcarnitine (C2) (acetylcarnitine);
    模型2方程为：ln[P/(1-P)]＝-0.06×V1-0.68×V2+0.83；其中，V1-V2顺次为：Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)，Acetylcarnitine(C2)(乙酰肉碱)；Model 2 equation is: ln[P/(1-P)]=-0.06×V1-0.68×V2+0.83; wherein, V1-V2 is: Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (androsterone sulfate Ketone/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Cholesterone Sulfate), Acetylcarnitine (C2) (acetylcarnitine);
    模型3方程为：ln[P/(1-P)]＝0.08×V1-0.18×V2-0.29×V3-0.49×V4-0.1.25×V5+2.08；其中V1-V5顺次为：Acrylic acid(丙烯酸)，3-Indoleacrylic acid(3-吲哚乙酸)，Ergothioneine(麦角硫因)，Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮),Acetylcarnitine(C2)(乙酰肉碱)；The equation of model 3 is: ln[P/(1-P)]＝0.08×V1-0.18×V2-0.29×V3-0.49×V4-0.1.25×V5+2.08; where V1-V5 is in sequence: Acrylic acid (acrylic acid), 3-Indoleacrylic acid (3-indole acetic acid), Ergothioneine (ergothioneine), Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (androsterone sulfate/dihydrotestosterone sulfate/epiandrosterone sulfate/sulfate This cholanolone), Acetylcarnitine (C2) (acetylcarnitine);
    模型4方程为：ln[P/(1-P)]＝1.06×V1+0.65×V2-0.51×V3-1.06×V4-1.11×V5-1.26×V6-1.26×V7-1.42×V8+4.8；其中，V1-V8顺次为：Acrylic acid(丙烯酸)，3-hydroxyoctanoylcarnitine(C8-OH)(3-羟基辛酰肉碱)，3-Indoleacrylic acid(3-吲哚乙酸)，Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)，Ergothioneine(麦角硫因)，Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)，Acetylcarnitine(C2)(乙酰肉碱)，Carnitine(肉碱)；或者Model 4 equation is: ln[P/(1-P)]=1.06×V1+0.65×V2-0.51×V3-1.06×V4-1.11×V5-1.26×V6-1.26×V7-1.42×V8+4.8; Among them, V1-V8 are: Acrylic acid (acrylic acid), 3-hydroxyoctanoylcarnitine (C8-OH) (3-hydroxyoctanoylcarnitine), 3-Indoleacrylic acid (3-indole acetic acid), Tetradecenoylcarnitine (C14:1 ) (5-myristoylcarnitine), Ergothioneine (ergothioneine), Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate (androsterone sulfate/dihydrotestosterone sulfate/epiandrosterone sulfate/bencholanolone sulfate) , Acetylcarnitine (C2) (acetylcarnitine), Carnitine (carnitine); or
    模型5方程为：ln[P/(1-P)]＝1.17×V1+0.84×V2+0.29×V3+0.06×V4-0.56×V5-1.26×V6-1.39×V7-1.4×V8-1.4×V9-1.69×V10+5.31，其中，V1-V10顺次为：Acrylicacid(丙烯酸)，3-hydroxyoctanoylcarnitine(C8-OH)(3-羟基辛酰肉碱)，3-hydroxydecanoylcarnitine(C10-OH)(3-羟基癸酰肉碱)，3-hydroxybutyrylcarnitine(C4-OH)(3-羟基丁酰肉碱)，Indole-3-acrylic acid(3-吲哚乙酸)，Ergothioneine(麦角硫因)，Acetylcarnitine(C2)(乙酰肉碱)，Androsterone sulfate/Dihydrotestosterone sulfate/Epiandrosterone sulfate/Etiocholanolone sulfate(硫酸雄酮/硫酸二氢睾酮/硫酸表雄酮/硫酸本胆烷醇酮)，Tetradecenoylcarnitine(C14:1)(5-豆蔻酰肉碱)，Carnitine(肉碱)。The equation of model 5 is: ln[P/(1-P)]＝1.17×V1+0.84×V2+0.29×V3+0.06×V4-0.56×V5-1.26×V6-1.39×V7-1.4×V8-1.4× V9-1.69×V10+5.31, wherein, V1-V10 is: Acrylicacid (acrylic acid), 3-hydroxyoctanoylcarnitine (C8-OH) (3-hydroxyoctanoylcarnitine), 3-hydroxydecanoylcarnitine (C10-OH) (3 -Hydroxydecanoylcarnitine), 3-hydroxybutyrylcarnitine (C4-OH) (3-hydroxybutyrylcarnitine), Indole-3-acrylic acid (3-indole acetic acid), Ergothioneine (ergothioneine), Acetylcarnitine (C2 ) (Acetylcarnitine), Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Etiocholanolone Sulfate (Androsterone Sulfate/Dihydrotestosterone Sulfate/Epiandrosterone Sulfate/Bencholanolone Sulfate), Tetradecenoylcarnitine(C14:1)(5- Myristoyl Carnitine), Carnitine (Carnitine).
  8. 根据权利要求7所述的***，该***还包括输入模块，就是输入所述标志物标志物的含量。The system according to claim 7, further comprising an input module for inputting the content of the marker marker.
  9. 根据权利要求8所述的***，当采用液相色谱-高分辨质谱联用检测以上标志物的时候，输入的是相对丰度值。According to the system according to claim 8, when the liquid chromatography-high resolution mass spectrometry is used to detect the above markers, the input is the relative abundance value.
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