CN113567585A - A peripheral blood-based screening marker and kit for esophageal squamous cell carcinoma - Google Patents
A peripheral blood-based screening marker and kit for esophageal squamous cell carcinoma Download PDFInfo
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Abstract
本发明属于医药生物技术领域，具体公开了一种食管鳞癌诊断的标志物及试剂盒。本发明提供的用于食管鳞癌诊断的标志物为S‑磺基‑L半胱氨酸、对称性二甲基精氨酸、4‑甲氧基苯乙酸中的至少一种，该标志物的检测试剂能够用于制备食管鳞癌诊断产品。本发明还提供一种用于食管鳞癌诊断的试剂盒，该试剂盒含有检测上述标志物的检测试剂，检测试剂为通过色谱法质谱法或色谱‑质谱联用法检测样本中的所述标志物的试剂。本发明通过检测人血清中S‑磺基‑L半胱氨酸、对称性二甲基精氨酸、4‑甲氧基苯乙酸的表达水平，可以有效检测食管鳞癌，其检测灵敏度高达80%，特异性达到71%，可用于食管鳞癌高发区无症状人群的大规模筛查，有利于无症状高危人群筛查和早期发现。The invention belongs to the field of medical biotechnology, and specifically discloses a marker and a kit for diagnosing esophageal squamous cell carcinoma. The marker for esophageal squamous cell carcinoma diagnosis provided by the present invention is at least one of S-sulfo-L cysteine, symmetrical dimethylarginine, and 4-methoxyphenylacetic acid, and the marker The detection reagent can be used to prepare esophageal squamous cell carcinoma diagnostic products. The present invention also provides a kit for diagnosing esophageal squamous cell carcinoma, the kit contains a detection reagent for detecting the above-mentioned marker, and the detection reagent is to detect the marker in a sample by chromatography-mass spectrometry or chromatography-mass spectrometry combined method reagent. The invention can effectively detect esophageal squamous cell carcinoma by detecting the expression levels of S-sulfo-L cysteine, symmetrical dimethylarginine and 4-methoxyphenylacetic acid in human serum, and the detection sensitivity is as high as 80 %, with a specificity of 71%, which can be used for large-scale screening of asymptomatic people in high-risk areas of esophageal squamous cell carcinoma, which is beneficial to the screening and early detection of asymptomatic high-risk groups.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly discloses a peripheral blood-based esophageal squamous carcinoma screening marker and a kit.
Background
Esophageal squamous carcinoma is one of the most common digestive system malignancies worldwide. According to the latest data of the world health organization (WH0) in 2018, 57 ten thousands of new esophageal squamous cell carcinoma cases are globally sent every year, about 51 thousands of cases die, and the seventh tumor incidence and the sixth mortality rate are located worldwide. According to analysis on the morbidity and mortality of malignant tumors in different areas of China in 2015 statistically released by the national cancer center, new cases of esophageal squamous cell carcinoma in China account for half of the world.
Because esophageal squamous carcinoma is very hidden in onset and extremely difficult to find and diagnose in early stage, most of patients who are first diagnosed clinically are in middle and late stages, so that the treatment prognosis is not ideal and the 5-year survival rate is low. With the increasing population base and the prolonging of the expected life of esophageal squamous cell carcinoma, a great number of people suffering from esophageal squamous cell carcinoma and death will bring serious burden to China in a long period of time in the future, and the method is the key point of tumor prevention and control.
At present, gastroscopy is the most effective examination for clinically diagnosing esophageal squamous carcinoma, and the development of various gastroscopy technologies makes important contribution to early diagnosis of esophageal squamous carcinoma. However, gastroscope census has many limitations, one is that the requirement on the operation experience of gastroscope doctors is high; the tolerance of the examined person is poor; secondly, the phenomenon of blindness and irregularity of gastroscope biopsy operation exists in asymptomatic people; thirdly, the detection rate of esophageal squamous carcinoma and various precancerous lesions in screening of asymptomatic population of esophageal squamous carcinoma high-incidence area by gastroscope is low, and the gastroscope examination is difficult to be carried out in large scale in the asymptomatic population of esophageal squamous carcinoma high-incidence area due to the reasons. If a noninvasive detection means for esophageal squamous cell carcinoma can be found, the molecular target with higher specificity and sensitivity for predicting the risk of esophageal squamous cell carcinoma has very important significance for early detection of esophageal squamous cell carcinoma, early warning and accurate screening of high-risk groups.
Esophageal squamous carcinogenesis is a complex multifactorial, multi-stage process, and the exact pathogenesis of esophageal squamous carcinogenesis is not clear. However, the obvious regional distribution (absolute high incidence of esophageal squamous cell carcinoma in Taihang mountain areas bordering the three provinces of Henan, Shanxi and Hebei) and the obvious family aggregation occurrence (40 percent of patients with positive family history) are the prominent epidemiological characteristics of the esophageal squamous cell carcinoma, and prompt that environmental and genetic factors play important roles in the esophageal squamous cell carcinoma. However, it has been found that individuals with different genetic backgrounds have different susceptibility to esophageal squamous cell carcinoma under the same environmental exposure. Metabolomics (metablomics/metablomics) is an emerging discipline that rapidly rises after genomics, transcriptomics, proteomics, and has become an important component of system biology. The method is a science for researching the change rule of the whole machine of endogenous metabolites generated after a biological system is stimulated or disturbed by outside, and is a research method for exploring the metabolic pathway of the biological system. The research object of metabonomics is small molecular substances with the relative molecular mass of below 1000Da, including: saccharides, amino acids, lipids, organic acids, carnitine, etc.
The organism in a normal state is a complete system, and metabolites in biological fluids, cells and tissues are in a stable equilibrium state. The organism is pathologically changed due to heredity or acquired reasons, the balance is broken, and metabolic products and metabolic processes are correspondingly changed. In the process of generating and developing tumors, tumor cells are stimulated by the external environment, and the tiny change of the internal environment can cause the 'delay effect' of metabolites, so that the metabolites are changed unusually. The change of the metabolic small molecules in the disease process is known through metabonomic analysis, so that people can be helped to search a related biomarker (biomar), can assist in the diagnosis of diseases, and can also be helped to know the pathogenesis of the diseases through the metabolic pathway related to the small molecule substances and provide a specific target for drug research and development. Since the advent of metabolomics, which has attracted great interest from scientists of various countries, has rapidly developed over a decade. Metabonomics has great potential in the fields of pathological elucidation, drug design and development, disease diagnosis, typing, disease prognosis and the like.
Metabolomics is a branch of system biology and has close relationship with genomics, transcriptomics and proteomics. It refers to the science of quantitatively measuring the composition of all metabolites of biological systems (cell models, tissues, organs or whole organisms), usually small molecule metabolites with relative molecular mass below 1000 daltons (Da), and the dynamic changes of these metabolites under external stimuli, using highly sensitive, high-throughput analytical techniques. Metabolomics is a high throughput method that can both comprehensively evaluate metabolites and qualitatively and quantitatively determine metabolites and analyze relevant metabolic pathways. Compared with other omics, the characteristics of integrity, macroscopicity and high throughput enable metabolomics to have incomparable advantages in biomarker screening and application, for example, the structure of a metabolome is much simpler than that of a proteome and a genome, and small changes of gene and protein expression can be amplified on a metabolite level and are easily detected. Therefore, the metabolite difference between the disease group and the control group is compared through metabonomic detection, so that the potential marker related to the disease is preliminarily determined, and the early diagnosis of clinical diseases is assisted. More and more studies have shown that using metabolomics methods, it is possible to make a diagnosis before the phenotype of the tumor cells changes, i.e. when the concentration of metabolites changes slightly. For quantitative and qualitative detection of metabolites, reliable and stable metabonomic detection techniques need to be resorted to. Since Warburg hypothesizes the specificity of metabolism to cancer cells due to altered mitochondrial defects, metabolic biomarkers have received attention from researchers as being effective biomarkers for early cancer diagnosis and prognosis. Since then, much effort has been devoted to the identification of metabolic biomarkers in oncology. Until now only two metabolic biomarkers have entered clinical practice, methoxy-adrenaline and methoxy-noradrenaline, respectively, as markers for predicting pheochromocytoma disease status. The key of metabonomics research lies in the rapid and accurate analysis and identification of a large number of small molecule metabolites, which depends on the progress of related technologies to a great extent. The development and combination of technologies such as nuclear magnetic resonance, mass spectrometry, chromatography and the like enable the wide application of metabonomics to be possible.
Currently, techniques for detecting and analyzing metabolites at the global level include liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and Nuclear Magnetic Resonance (NMR). The NMR is characterized by no damage to the components to be detected, simple sample pretreatment, low sensitivity and narrow detection dynamic range; GC-MS has good sensitivity and reproducibility, but a derivatization method is generally adopted to carry out pretreatment on a sample, so that the experimental steps become complicated. Compared with GC-MS, LC-MS can analyze compounds with high polarity and relatively higher molecular weight, and is more suitable for detecting complex metabolites in metabonomic biological samples and identifying potential markers. In addition, the method has the characteristics of simple sample treatment, high sensitivity and strong clinical practicability, so that the method adopts LC-MS to carry out metabonomics analysis on the metabolic small molecules, if stable specific plasma metabolic small molecules related to the onset of the esophageal squamous cell carcinoma are found to be used as biomarkers, and an LC-MS detection method for metabolic small molecule markers of corresponding diseases is developed, the method is in an international leading position in the field, can create remarkable economic benefits, and can be a powerful promotion for early discovery and early diagnosis of esophageal squamous cell carcinoma in China.
Disclosure of Invention
Aiming at the problems and the defects in the prior art, one object of the invention is to provide a marker for screening esophageal squamous carcinoma based on peripheral blood, the other object of the invention is to provide an application of a detection reagent of the marker for screening esophageal squamous carcinoma in the preparation of products for screening esophageal squamous carcinoma, and the third object of the invention is to provide a kit for screening esophageal squamous carcinoma.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
the invention provides a marker for diagnosing esophageal squamous carcinoma, wherein the marker is at least one of S-Sulfo-L cysteine (S-Sulfo-L-cysteine), Symmetric Dimethylarginine (SDMA) and 4-methoxyphenylacetic acid.
Preferably, the marker is a serum marker according to the above.
In a second aspect, the invention provides a use of the detection reagent for the marker of the first aspect in the preparation of a product for diagnosing esophageal squamous cell carcinoma.
According to the above-mentioned application, preferably, the test sample of the product is serum.
According to the above-mentioned use, preferably, the detection reagent is a reagent for detecting the marker in the sample by chromatography, mass spectrometry or a combination of chromatography and mass spectrometry.
According to the above-mentioned use, preferably, the chromatography is gas chromatography, liquid chromatography or high performance liquid chromatography.
According to the above application, preferably, the chromatography-mass spectrometry combination is a gas chromatography-mass spectrometry combination, a liquid chromatography-mass spectrometry combination, a high performance liquid chromatography-mass spectrometry combination, a gas chromatography-tandem mass spectrometry combination, a liquid chromatography-tandem mass spectrometry combination or a high performance liquid chromatography-tandem mass spectrometry combination.
According to a third aspect of the present invention, there is provided a kit for diagnosing esophageal squamous carcinoma, the kit comprising a detection reagent for detecting the marker of the first aspect, wherein the marker is at least one of S-sulfo-L cysteine, symmetric dimethylarginine and 4-methoxyphenylacetic acid.
According to the above kit, preferably, the detection reagent is a reagent for detecting the marker in the sample by chromatography-mass spectrometry or a chromatography-mass spectrometry combination.
Preferably, the chromatography is gas chromatography, liquid chromatography or high performance liquid chromatography according to the above-mentioned kit; the chromatography-mass spectrometry combination method is a gas chromatography-mass spectrometry combination method, a liquid chromatography-mass spectrometry combination method, a high performance liquid chromatography-mass spectrometry combination method, a gas chromatography-tandem mass spectrometry combination method, a liquid chromatography-tandem mass spectrometry combination method or a high performance liquid chromatography-tandem mass spectrometry combination method.
Preferably, the kit further comprises a standard for the marker of the first aspect.
According to the kit, preferably, the detection sample of the kit is serum.
In the invention, the esophageal squamous carcinoma specifically refers to esophageal squamous carcinoma.
Compared with the prior art, the invention has the following positive beneficial effects:
(1) the invention discovers for the first time that three substances, namely S-sulfo-L cysteine, symmetrical dimethyl arginine and 4-methoxyphenylacetic acid, can be used for diagnosing and detecting esophageal squamous carcinoma by a metabonomics method, and can effectively detect the esophageal squamous carcinoma, particularly early esophageal squamous carcinoma by detecting the expression levels of the S-sulfo-L cysteine, the symmetrical dimethyl arginine and the 4-methoxyphenylacetic acid in human serum; proved by verification, when any marker of S-sulfo-L cysteine, symmetrical dimethylarginine and 4-methoxyphenylacetic acid is singly adopted for carrying out esophageal squamous carcinoma screening, the AUC value of an ROC curve is over 0.6; when a plurality of markers are used in a combined mode, the AUC value of the ROC curve is closer to 1 than that of a single index, the distinguishing effect is good, and the diagnosis effect is good. Therefore, the marker for screening esophageal squamous carcinoma can be used for early screening of esophageal squamous carcinoma.
(2) The invention uses the three markers of S-sulfo-L cysteine, symmetrical dimethylarginine and 4-methoxyphenylacetic acid as a combination for screening and detecting early esophageal squamous carcinoma, the detection sensitivity is as high as 80 percent (namely the rate of correctly diagnosing early esophageal squamous carcinoma when the three markers are applied to early esophageal squamous carcinoma patients for diagnosis is 80 percent), the specificity is as high as 71 percent (namely the rate of determining people without esophageal squamous carcinoma when the three markers are applied to non-esophageal squamous carcinoma patients for diagnosis is 71 percent), therefore, the marker of the invention has higher sensitivity and specificity, greatly improves the detection rate of early esophageal squamous carcinoma, and the detection rate of esophageal squamous carcinoma is far higher than that of screening esophageal squamous carcinoma by the existing clinical endoscope (2 to 3 percent), can be used for large-scale screening of people with asymptomatic high risk in esophageal squamous carcinoma high-incidence areas, meanwhile, an important detection means is provided for realizing long-term tracking of asymptomatic high-risk groups in an esophageal squamous carcinoma high-risk area, early discovery of the asymptomatic esophageal squamous carcinoma high-risk groups is facilitated, the death rate of patients suffering from esophageal squamous carcinoma is greatly reduced, and great welfare is brought to the patients suffering from esophageal squamous carcinoma and families.
(3) The marker for screening the esophageal squamous cell carcinoma is a serum detection marker, so that invasive diagnosis can be avoided, and the risk of the esophageal squamous cell carcinoma can be obtained at an early stage by taking serum for detection in a minimally invasive way, so that a basis is provided for further and deep inspection by a clinician, support is provided for rapidly and accurately mastering the disease state and the severity of the condition of a patient, a more personalized prevention and treatment scheme is adopted in time, and the disease progress is delayed and prevented.
(4) The detection sample of the kit for screening esophageal squamous carcinoma is serum, so that the blood demand is low, the pain of the masses is low, and the acceptance is high; moreover, the method is simple to operate, short in detection result time and wide in market prospect and social benefit.
Drawings
FIG. 1 is a ROC plot using S-sulfo-L cysteine diagnosis to differentiate esophageal squamous carcinoma patient groups from normal control groups;
FIG. 2 is a ROC plot using symmetric dimethylarginine diagnosis to differentiate esophageal squamous carcinoma patient groups from normal control groups;
FIG. 3 is a ROC graph for distinguishing an esophageal squamous carcinoma patient group from a normal control group by using 4-methoxyphenylacetic acid diagnosis;
FIG. 4 is a ROC plot using S-sulfo-L-cysteine in combination with symmetric dimethylarginine for diagnosis of patients with esophageal squamous carcinoma differentiated from normal controls;
FIG. 5 is a ROC plot for the discrimination of esophageal squamous carcinoma patient groups from normal control groups using S-sulfo-L-cysteine in combination with 4-methoxyphenylacetic acid;
FIG. 6 is a ROC plot for the discrimination of esophageal squamous carcinoma patient groups from normal control groups using a combination of symmetric dimethylarginine and 4-methoxyphenylacetic acid;
FIG. 7 is a ROC plot for the discrimination of esophageal squamous carcinoma patient groups from normal control groups using S-sulfo-L-cysteine, symmetric dimethylarginine, and 4-methoxyphenylacetic acid in combination.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed description and accompanying drawings. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention.
The experimental procedures described in the following examples, unless otherwise specified, are conventional in the art or according to the conditions recommended by the manufacturers; the reagents, materials and instruments used are not indicated by manufacturers, and are all conventional products commercially available.
Example 1: screening of esophageal squamous carcinoma serum differential metabolic markers
1. Experimental sample
100 healthy subjects (normal control group) and 100 patients with esophageal squamous carcinoma (esophageal squamous carcinoma group) aged and sex-matched at the first subsidiary hospital of zhengzhou university were collected according to strict screening and exclusion criteria. Average age of 100 healthy subjects: age 58.5 years, range 42-80 years; average age of 100 patients with esophageal squamous carcinoma: 59.5 years old, range 42-83 years old.
The inclusion criteria for healthy subjects were: age between 42 and 80 years; the tumor is not diagnosed by physical examination, and the history of the tumor disease does not exist; no other systemic major diseases; there is no history of chronic disease with long-term medication.
The grouping standard of esophageal squamous carcinoma patients is as follows: age between 42 and 83 years; patients with esophageal squamous carcinoma who are determined by endoscopy and confirmed by histopathology do not receive radiotherapy or chemotherapy treatment; no other systemic major diseases; there is no history of chronic disease with long-term medication.
2. Experimental methods
(1) Collecting and storing serum samples:
collecting fasting peripheral blood of a patient in the early morning, placing the fasting peripheral blood in a test tube without anticoagulant, naturally coagulating for 30-60min at room temperature, after blood coagulation, centrifuging for 10min at 2000rpm, carefully sucking supernatant clear serum liquid into a sterile freeze-drying tube, marking, and storing in a refrigerator at-80 ℃ for later use.
(2) The main reagents are as follows:
methanol and acetonitrile (UPLC pure) from merk, usa, and chromatographic grade formic acid and ammonium acetate from ROE, usa; deionized water was prepared from the Milli-Q ultrapure water system from Millipore, Inc., USA; standards include S-sulfo-L cysteine, symmetric dimethylarginine, 4-methoxyphenylacetic acid available from Sigma-Aldrich, USA. The internal standard L-2 chlorophenylalanine was purchased from Shanghai Michelin Biochemical technology Ltd, and ketoprofen was purchased from the Chinese food and drug testing institute.
(3) UPLC-Q/TOF-MS detection:
A) a detection instrument:
a Waters Acquity UPLC ultra-high performance liquid chromatograph and an Agilent 6545 quadrupole-time-of-flight mass spectrometer. .
B) Chromatographic conditions are as follows:
a chromatographic column: an Acquity BEH C18 analytical column (100X 2.1mm,1.7 μm, Waters, USA) was used, the column temperature was 40 ℃, the flow rate was 0.4ml/min, the sample size was 5ul, and the sample injection was repeated three times for each sample. The mobile phase composition is as follows: phase A is 0.2% formic acid water solution; the phase B is pure acetonitrile, a sample is eluted in a gradient mode, and the mobile phase composition is 0-3min and 5% -70% of phase B; 3-8min, 90% B; 8-9min, 100% B; 9-10min, 5% -100% B; 10-12min, 5% B.
C) Mass spectrum conditions:
the mass spectrum collection adopts a positive ion V mode, and the detection parameters are set as follows: capillary voltage 3.2kV, taper hole voltage 30V, desolvation gas temperature 350 ℃, ion source temperature 120 ℃, desolvation gas flow 700L/H, taper hole gas flow 40L/H, acquisition time range 0-12min, scanning range 50-1100M/z, scanning time 0.2s each time, scanning interval time 0.02s for two times, locking mass calibration mode DRE, and performing real-time accurate mass calibration [ (M + H) with leucine-enkephalin+＝556.2771]。
D) Sample treatment:
taking 50 mu L of serum sample, adding 150 mu L of methanol with the volume being 3 times that of the serum sample, vortexing for 30s, uniformly mixing, putting into a high-speed centrifuge for centrifugation, and centrifuging for 10min at 13000 rpm. And sucking 75 mu L of centrifuged supernatant, respectively putting the supernatant into 2 centrifuge tubes with the volume of 1.5mL, drying the supernatant by using a nitrogen blower, re-dissolving the supernatant by respectively using 100 mu L of methanol complex solution containing an internal standard solution (L-2-chlorophenylalanine) or 100 mu L of methanol complex solution containing an internal standard solution (ketoprofen) after drying, wherein the final concentrations of the L-2-chlorophenylalanine and the ketoprofen are respectively 100ng/mL and 1 mu g/mL, and the final concentrations are respectively used as positive and negative ion mode detection samples. And (3) after redissolving, uniformly mixing by vortexing for 30s, putting the mixture into a high-speed centrifuge for centrifugation at 13000rpm for 10min, sucking the centrifuged supernatant, and then putting the supernatant into a liquid vial for UPLC-Q/TOF-MS detection.
(4) Data processing method
A) Data preprocessing
Based on data obtained by UPLC-Q/TOF-MS, under an R software platform, XCMS program codes are adopted for extracting peaks, aligning and deconvoluting analyzing, and screening the peaks according to the principle of 80% is carried out to obtain a three-dimensional visual matrix containing variables (retention time Rt, mass-to-charge ratio m/z), observation and peak intensity, and the data matrix is imported into SIMCA-P software (version 13.0) for multivariate statistical analysis.
B) Multivariate statistical analysis
In order to examine the metabolic change of the esophageal squamous cell carcinoma group compared with a normal control group, firstly, unsupervised Principal Component Analysis (PCA) is adopted for all variables, the clustering condition of each group of data is observed, the outlier is removed, finally, supervised data analysis is carried out by adopting a least square method of orthogonal analysis (PLS-DA) model, and the difference among groups is amplified so as to obtain the most obvious separation among groups.
C) And detecting excavation and identification of differential metabolites between an esophageal squamous carcinoma group and a normal control group by using UPLC-Q/TOF-MS:
by combining the VIP values under the PLS-DA models of the esophageal squamous carcinoma group and the normal control group with the P value of the single-factor statistical analysis, the variables with VIP >1.0 and P <0.05 are considered to have significant difference, and the variables with significant difference are considered to be the differential metabolism biomarkers.
The selected differential variable needs to be assigned to the biomarker it represents. Metabolite identification based on UPLC-Q/TOF-MS technology is mainly by matching via a library of metabolite profiles: finding mass spectrograms of differential variables on a UPLC-Q/TOF-MS total ion flow graph, comparing the precise molecular weights of the differential metabolites with a network database, such as HMDB (http:// www.hmdb.ca), METLIN (http:// METLIN. script. edu) and KEGG (http:// www.kegg.jp), preliminarily identifying the structures of the differential metabolites, finally determining the structures of the differential metabolites by purchasing standard products, comparing the molecular weights, chromatographic retention times and corresponding multi-stage MS cracking spectra of the standard products, preparing a series of standard product solutions with known concentrations, and further determining the content of the differential metabolites through a standard curve.
3. Results of the experiment
According to the experimental method, different metabolites among groups are screened by combining the VIP value in PLS-DA and the t test result, and 9 metabolites with difference among the esophageal squamous carcinoma group and the normal control group are obtained through retrieval and identification of a Human Metabolome Database (HMDB), wherein the relative concentration difference of the S-sulfo-L cysteine, the symmetric dimethylarginine and the 4-methoxyphenylacetic acid has statistical significance (P <0.05), and the metabolites are different metabolites with significant difference, and are shown in Table 1. Moreover, compared with a normal control group, the relative contents of S-sulfo-L cysteine, symmetrical dimethylarginine and 4-methoxyphenylacetic acid in the serum of the patient with esophageal squamous carcinoma are obviously increased.
TABLE 1 statistic results of metabolites in esophageal squamous carcinoma detection group and normal control group based on UPLC-Q/TOF-MS
| Differential metabolites | p | VIP | Ratio of |
| S-sulfo-L-cysteine | 0.001 | 2.582 | ＞1 |
| Symmetrical dimethylarginine | 0.002 | 2.514 | ＞1 |
| 4-Methoxyphenylacetic acid | 0.03 | 2.272 | ＞1 |
Note: the ratio indicates the relative level of the compound in the esophageal squamous carcinoma test group, wherein >1 indicates an increase and <1 indicates a decrease.
Example 2: assessment of the ability of differential metabolite diagnosis to differentiate esophageal squamous carcinoma patients from healthy persons
1. Single differential metabolite diagnosis the ability to distinguish esophageal squamous carcinoma patients from normal:
the ability of each differential metabolite to differentiate esophageal squamous cell carcinoma patients from normal individuals by individual diagnosis was evaluated using a receiver operating curve (ROC curve) based on the analysis of data on the levels of S-sulfo-L-cysteine, symmetric dimethylarginine, 4-methoxyphenylacetic acid in serum samples from the esophageal squamous cell carcinoma group (100 patients with esophageal squamous cell carcinoma) and a normal control group (100 healthy subjects) as measured by UPLC-Q/TOF-MS in example 1. The ROC curves for the individual diagnosis of S-sulfo-L cysteine, symmetric dimethylarginine and 4-methoxyphenylacetic acid to distinguish patients with esophageal squamous carcinoma from normal persons are shown in FIG. 1, FIG. 2 and FIG. 3. According to the ROC curve, the area under the curve, AUC, sensitivity and specificity of the ROC curve of each differential metabolite are calculated, and the results are shown in Table 2.
TABLE 2 AUC for the differentiation of esophageal squamous carcinoma patients from normal human by the independent diagnosis of three differential metabolites
| Numbering | Differential metabolites | AUC | Sensitivity of the probe | Degree of specificity |
| 1 | S-sulfo-L-cysteine | 0.679 | 43.0％ | 95.0％ |
| 2 | Symmetrical dimethylarginine | 0.617 | 40.0％ | 91.0％ |
| 3 | 4-Methoxyphenylacetic acid | 0.620 | 35.0％ | 93.0％ |
The area AUC under the ROC curve is generally accepted as the inherent accuracy index of the authenticity evaluation of the diagnostic test, and when the AUC is 0.5, the diagnostic significance is not achieved; when the AUC is 0.5-0.7, the diagnosis accuracy is low; when the AUC is 0.7-0.9, the diagnosis accuracy is moderate; AUC > 0.9, indicating higher accuracy of diagnosis. As can be seen from Table 2 and FIGS. 1, 2 and 3, the AUC of the ROC curve of the individual markers S-sulfo-L cysteine, symmetric dimethylarginine and 4-methoxyphenylacetic acid for distinguishing patients with esophageal squamous carcinoma from the normal persons can reach over 0.6, which indicates that S-sulfo-L cysteine, symmetric dimethylarginine or 4-methoxyphenylacetic acid can be used for diagnosing and distinguishing patients with esophageal squamous carcinoma from the normal persons.
Further, a jotan index (sensitivity + specificity-1) was calculated from the coordinates of the ROC curve, and the relative metabolite content at the maximum jotan index was the optimal cutoff value for diagnosis and differentiation of patients with esophageal squamous cell carcinoma from normal persons, as shown in table 3.
TABLE 3 john's index and cutoff for the individual diagnosis of three differential metabolites to differentiate patients with esophageal squamous carcinoma from normal
| Numbering | Differential metabolites | Joden index | Optimum cutoff value |
| 1 | S-sulfo-L-cysteine | 0.38 | 0.567 |
| 2 | Symmetrical dimethylarginine | 0.31 | 0.551 |
| 3 | 4-Methoxyphenylacetic acid | 0.28 | 0.675 |
2. Multiple differential metabolite combination diagnostics ability to distinguish esophageal squamous carcinoma patients from normal:
(1) S-sulfo-L cysteine and symmetric dimethylarginine in combination to differentiate esophageal squamous carcinoma patients from normal:
relative amounts of S-sulfo-L-cysteine and symmetric dimethylarginine in the serum samples of the esophageal squamous carcinoma group (100 patients with esophageal squamous carcinoma) and the normal control group (100 healthy subjects) tested by UPLC-Q/TOF-MS in example 1 were used as independent variables (let X be1(S-sulfo-L-cysteine)Relative content of acid, X2Relative content of symmetric dimethylarginine), taking the group (esophageal squamous carcinoma group and normal control group) as dependent variable, and performing binary logistic regression on the relative contents of S-sulfo-L cysteine and symmetric dimethylarginine in the serum samples of the esophageal squamous carcinoma group and the normal control group to obtain a binary logistic regression equation: logit [ p ]]＝-2.168+3.235X1+2.299X2(ii) a Substituting the relative contents of S-sulfo-L cysteine and symmetrical dimethylarginine in each serum sample into the binary logistic regression equation to obtain the regression value logit [ p ] of each serum sample]With possible regression values logit [ p ]]As a diagnosis point, the sensitivity and specificity were calculated, and then an ROC curve was plotted based on the calculated sensitivity and specificity, the ROC curve being shown in FIG. 4.
According to the ROC curve, the area AUC under the ROC curve for distinguishing esophageal squamous carcinoma patients from normal people in the combined diagnosis of S-sulfo-L cysteine and symmetric dimethylarginine is 0.747, and the method has higher accuracy. And further calculating a jotan index (the jotan index is sensitivity + specificity-1) according to the coordinates of the ROC curve, wherein the corresponding logit [ p ] value when the jotan index is maximum is the optimal cut-off value for diagnosing and distinguishing esophageal squamous cell carcinoma patients from normal people, and the optimal cut-off value is 0.051.
(2) S-sulfo-L-cysteine and 4-methoxyphenylacetic acid in combination with a diagnostic agent to differentiate patients with esophageal squamous carcinoma from normal persons:
relative amounts of S-sulfo-L-cysteine and 4-methoxyphenylacetic acid in the serum samples of the esophageal squamous carcinoma group (100 patients with esophageal squamous carcinoma) and the normal control group (100 healthy subjects) tested by UPLC-Q/TOF-MS in example 1 were used as independent variables (let X be1Relative content of S-sulfo-L-cysteine, X24-methoxyphenylacetic acid relative content), taking the group (esophageal squamous carcinoma group and normal control group) as a dependent variable, and performing binary logistic regression on the relative contents of S-sulfo-L cysteine and 4-methoxyphenylacetic acid in serum samples of the esophageal squamous carcinoma group and the normal control group to obtain a binary logistic regression equation: logit [ p ]]＝-2.264+3.293X1+2.247X2(ii) a The relative contents of S-sulfo-L cysteine and 4-methoxyphenylacetic acid in each serum sample were determinedSubstituting into the binary logistic regression equation to obtain the regression value logit [ p ] of each serum sample]With possible regression values logit [ p ]]As a diagnosis point, the sensitivity and specificity were calculated, and then an ROC curve was plotted based on the calculated sensitivity and specificity, as shown in FIG. 5.
According to the ROC curve, the area AUC under the ROC curve for distinguishing the esophageal squamous carcinoma patients from normal people in the combined diagnosis of the S-sulfo-L cysteine and the 4-methoxyphenylacetic acid is 0.744, and the method has higher accuracy. And further calculating a jotan index (sensitivity + specificity-1) according to coordinates of the ROC curve, wherein the corresponding logit [ p ] value when the jotan index is maximum is the optimal cut-off value for diagnosing and distinguishing esophageal squamous cell carcinoma patients from normal people, and the optimal cut-off value is 0.398.
(3) The ability of the combined diagnosis of symmetric dimethylarginine and 4-methoxyphenylacetic acid to distinguish esophageal squamous carcinoma patients from normal persons:
relative amounts of symmetric dimethylarginine and 4-methoxyphenylacetic acid in serum samples of the esophageal squamous carcinoma group (100 esophageal squamous carcinoma patients) and the normal control group (100 healthy subjects) tested by UPLC-Q/TOF-MS in example 1 were used as independent variables (let X be set1Relative content of symmetric dimethylarginine, X2Relative content of 4-methoxyphenylacetic acid), taking the group (esophageal squamous carcinoma group and normal control group) as a dependent variable, and performing binary logistic regression on the relative contents of the symmetric dimethylarginine and the 4-methoxyphenylacetic acid in the serum samples of the esophageal squamous carcinoma group and the normal control group to obtain a binary logistic regression equation: logit [ p ]]＝-1.533+1.949X1+1.854X2(ii) a Substituting the relative contents of the symmetrical dimethylarginine and the 4-methoxyphenylacetic acid in each serum sample into the binary logistic regression equation to obtain the regression value logit [ p ] of each serum sample]With possible regression values logit [ p ]]As a diagnosis point, the sensitivity and specificity were calculated, and then an ROC curve was plotted based on the calculated sensitivity and specificity, as shown in FIG. 6.
According to the ROC curve, the area AUC under the ROC curve for distinguishing the esophageal squamous carcinoma patients from the normal people in the combined diagnosis of the symmetrical dimethylarginine and the 4-methoxyphenylacetic acid is 0.700, and the method has higher accuracy. And further calculating a jotan index (sensitivity + specificity-1) according to coordinates of the ROC curve, wherein the corresponding logit [ p ] value when the jotan index is maximum is the optimal cut-off value for diagnosing and distinguishing esophageal squamous cell carcinoma patients from normal people, and the optimal cut-off value is 0.068.
(4) S-sulfo-L-cysteine, symmetric dimethylarginine, and 4-methoxyphenylacetic acid in combination to diagnose the ability to differentiate esophageal squamous carcinoma patients from normal:
relative contents of S-sulfo-L cysteine, symmetric dimethylarginine and 4-methoxyphenylacetic acid in serum samples of the esophageal squamous carcinoma group (100 esophageal squamous carcinoma patients) and the normal control group (100 healthy subjects) detected by UPLC-Q/TOF-MS in example 1 were used as independent variables (X is set1Relative content of S-sulfo-L-cysteine, X2Relative content of symmetric dimethylarginine, X34-methoxyphenylacetic acid), taking the group (esophageal squamous carcinoma group and normal control group) as a dependent variable, and performing binary logistic regression on the relative contents of S-sulfo-L cysteine, symmetric dimethylarginine and 4-methoxyphenylacetic acid in serum samples of the esophageal squamous carcinoma group and the normal control group to obtain a binary logistic regression equation: logit [ p ]]＝-3.283+3.580X1+2.311X2+2.307X3(ii) a Substituting the relative contents of S-sulfo-L cysteine, symmetrical dimethylarginine and 4-methoxyphenylacetic acid in each serum sample into the binary logistic regression equation to obtain the regression value logit [ p ] of each serum sample]With possible regression values logit [ p ]]As a diagnosis point, the sensitivity and specificity were calculated, and then an ROC curve was plotted based on the calculated sensitivity and specificity, as shown in FIG. 7.
According to the ROC curve, the area AUC under the ROC curve for distinguishing patients with esophageal squamous cell carcinoma from normal people in the combined diagnosis of S-sulfo-L cysteine, symmetrical dimethylarginine and 4-methoxyphenylacetic acid is 0.800, and the method has high accuracy. And calculating a jotan index (sensitivity + specificity-1) according to the coordinates of the ROC curve, wherein the corresponding logit [ p ] value when the jotan index is maximum is the optimal cut-off value for diagnosing and distinguishing the esophageal squamous cell carcinoma patient from the normal person, and the optimal cut-off value is 0.209.
The combination diagnosis of single or multiple differential metabolites and the differentiation of the ROC curve AUC value, sensitivity, specificity, Yoden index and optimal cutoff value of the esophageal squamous cell carcinoma patients from normal people are counted, and the specific results are shown in Table 4.
TABLE 4 AUC values for different differential metabolite combinations for diagnostic differentiation of patients with esophageal squamous carcinoma from normal
As can be seen from Table 4, compared with a single differential metabolite, when the esophageal squamous carcinoma patients and the normal persons are diagnosed and distinguished by adopting the combination of any two markers of S-sulfo-L cysteine, symmetric dimethylarginine and 4-methoxyphenylacetic acid, the AUC of the ROC curve can reach more than 0.70, and the AUC is obviously higher than that of the single marker; when three different metabolites are used for combined diagnosis to distinguish esophageal squamous carcinoma patients from normal people, the AUC of the ROC curve reaches the maximum of 0.805, and the sensitivity of esophageal squamous carcinoma diagnosis also reaches the highest, which shows that the diagnosis effect is optimal when the three markers are combined. In addition, the jotan index is obtained by subtracting 1 from the sum of sensitivity and specificity in statistics, the numerical range is 0-1, and the closer the jotan index is to 1, the higher the diagnostic value is, and the higher the application value of the method is. With the increase of the number of marker combinations, the john index is continuously increased and gradually tends to 1, which indicates that the method for diagnosing esophageal squamous carcinoma by using 3 marker combinations has better diagnostic value.
Example 3: application of three differential metabolites in esophageal squamous carcinoma screening
1. Collection of serum samples
50 healthy subjects (normal control group) and 50 patients with esophageal squamous carcinoma (esophageal squamous carcinoma group) aged and sex-matched at the first subsidiary hospital of Zhengzhou university were collected according to strict screening and exclusion criteria.
The inclusion criteria for healthy subjects were: age between 42 and 80 years; the tumor is not diagnosed by physical examination, and the history of the tumor disease does not exist; no other systemic major diseases; there is no history of chronic disease with long-term medication.
The grouping standard of esophageal squamous carcinoma patients is as follows: age between 42 and 83 years; patients with esophageal squamous carcinoma who are determined by endoscopy and confirmed by histopathology do not receive radiotherapy or chemotherapy treatment; no other systemic major diseases; there is no history of chronic disease with long-term medication.
2. Experimental methods
(1) Collecting and storing serum samples:
collecting fasting peripheral blood of a patient in the early morning, placing the fasting peripheral blood in a test tube without anticoagulant, naturally coagulating for 30-60min at room temperature, after blood coagulation, centrifuging for 10min at 2000rpm, carefully sucking supernatant clear serum liquid into a sterile freeze-drying tube, marking, and storing in a refrigerator at-80 ℃ for later use.
3. Experimental and analytical methods
Qualitative and quantitative determination of the three metabolites (S-sulfo-L cysteine, symmetric dimethylarginine, 4-methoxyphenylacetic acid) was performed on the sera of 50 healthy subjects (normal control group) and 50 patients with esophageal squamous carcinoma (esophageal squamous carcinoma group) according to the UPLC-Q/TOF-MS experimental method described in example 1.
When S-sulfo-L cysteine, symmetrical dimethyl arginine or 4-methoxyphenylacetic acid is singly used for diagnosing the esophageal squamous cell carcinoma, the negative and the positive of the sample are judged according to the content of the S-sulfo-L cysteine, the symmetrical dimethyl arginine or the 4-methoxyphenylacetic acid in the serum sample and the optimal cutoff value of the corresponding differential metabolite calculated in the embodiment 2, and the esophageal squamous cell carcinoma patient is judged if the content of the differential metabolite in the serum sample is higher than the optimal cutoff value, otherwise, the normal person is judged.
When the combination of three metabolic markers of S-sulfo-L cysteine, symmetrical dimethyl arginine and 4-methoxyphenylacetic acid is used for diagnosing esophageal squamous carcinoma, the contents of S-sulfo-L cysteine, symmetrical dimethyl arginine and 4-methoxyphenylacetic acid in a serum sample are substituted into the Logistic regression equation obtained in the example 2, the optimal cutoff value of esophageal squamous carcinoma patients and normal persons is distinguished according to the calculated logit [ p ] value and the S-sulfo-L cysteine, symmetrical dimethyl arginine and 4-methoxyphenylacetic acid combined diagnosis obtained in the example 2, the negative and positive of the sample are judged, if the logit [ p ] value is higher than the optimal cutoff value, the esophageal squamous carcinoma is judged, otherwise, the normal persons are judged.
3. The result of the detection
TABLE 5 results of the diagnosis of esophageal squamous carcinoma by the combination of two metabolic markers
The results are shown in Table 5. As can be seen from Table 5, when S-sulfo-L cysteine, symmetric dimethylarginine or 4-methoxyphenylacetic acid is independently adopted for diagnosing esophageal squamous cell carcinoma, the positive predictive value and the negative predictive value can both reach more than 80 percent; when any two differential metabolites are combined for diagnosing esophageal squamous carcinoma, the positive predicted value and the negative predicted value of the differential metabolites are obviously higher than those of a single differential metabolite; moreover, when three differential metabolites of S-sulfo-L cysteine, symmetric dimethylarginine and 4-methoxyphenylacetic acid are combined for diagnosing the esophageal squamous cell carcinoma, the positive predicted value and the negative predicted value of the differential metabolites reach the highest values, namely 0.961 and 0.979 respectively. Therefore, the diagnosis effect of the esophageal squamous carcinoma is best by combining three different metabolites, namely S-sulfo-L cysteine, symmetric dimethylarginine and 4-methoxyphenylacetic acid.
Example 4: preparation of esophageal squamous carcinoma screening kit based on metabolic marker
Based on the 3 metabolic markers related to the esophageal squamous cell carcinoma obtained by screening, the esophageal squamous cell carcinoma screening kit is designed, and comprises the following components:
standard for markers: at least one of S-sulfo-L cysteine, symmetrical dimethylarginine and 4-methoxyphenylacetic acid, and the kit can contain one of the standard substances, or two of the standard substances, or 3 of the standard substances, and can be combined according to requirements. When more than one metabolic marker standard is involved, each marker standard may be packaged separately or each marker calibrator may be mixed to make a mixture package.
The application process of the kit comprises the following steps: collecting serum of a subject, freezing and storing the serum in a refrigerator at minus 80 ℃, unfreezing a serum sample in a refrigerator at 4 ℃ before an experiment, taking 50 mu L of the serum sample, then adding 150 mu L of methanol which is 3 times of the volume of the serum sample, uniformly mixing the serum sample by vortex for 30s, putting the mixture into a high-speed centrifuge for centrifugation after uniform mixing, and centrifuging the mixture for 10min at 13000 rpm. Sucking 75 mu L of centrifuged supernatant, respectively putting the supernatant into 2 centrifuge tubes with the volume of 1.5ml, blowing the supernatant to dry by using a nitrogen blowing instrument, redissolving by using 100 mu L of methanol containing an internal standard solution (L-2-chlorophenylalanine or ketoprofen), uniformly mixing by swirling for 30s after redissolution, putting the mixture into a high-speed centrifuge for centrifugation at 13000rpm for 10min, sucking the centrifuged supernatant, and then putting the supernatant into a liquid vial for UPLC-Q/TOF-MS detection.
The treated serum samples were analyzed according to the UPLC-MS instrument setup method of example 1 and the markers were quantitatively and qualitatively analyzed with reference to the data processing method of example 1.
When the esophageal squamous carcinoma detection kit is used, 3 markers are recommended to be detected simultaneously so as to further improve the detection efficiency.
The above-described embodiments are intended to illustrate the substance of the present invention, but are not intended to limit the scope of the present invention. It will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the invention.
Claims (10)
1. The marker for screening the esophageal squamous carcinoma is at least one of S-sulfo-L cysteine, symmetrical dimethylarginine and 4-methoxyphenylacetic acid.
2. Use of a detection reagent for the marker of claim 1 in the preparation of a product for esophageal squamous carcinoma screening.
3. The use according to claim 2, wherein the test sample of the product is serum.
4. The use of claim 2, wherein the detection reagent is a reagent for detecting the marker in the sample by chromatography, mass spectrometry or a combination of chromatography and mass spectrometry.
5. Use according to claim 4, wherein the chromatography is gas chromatography, liquid chromatography or high performance liquid chromatography.
6. The use of claim 4, wherein the chromatography-mass spectrometry combination is a gas chromatography-mass spectrometry combination, a liquid chromatography-mass spectrometry combination, a high performance liquid chromatography-mass spectrometry combination, a gas chromatography-tandem mass spectrometry combination, a liquid chromatography-tandem mass spectrometry combination, or a high performance liquid chromatography-tandem mass spectrometry combination.
7. A kit for esophageal squamous carcinoma screening, which comprises a detection reagent for detecting the marker of claim 1.
8. The kit of claim 7, wherein the detection reagent is a reagent for detecting the marker in the sample by chromatography-mass spectrometry or a combination of chromatography-mass spectrometry.
9. The kit of claim 8, wherein the chromatography is gas chromatography, liquid chromatography or high performance liquid chromatography; the chromatography-mass spectrometry combined method is a gas chromatography-mass spectrometry combined method, a liquid chromatography-mass spectrometry combined method or a high performance liquid chromatography-mass spectrometry combined method.
10. The kit of claim 9, further comprising a standard for the marker of claim 1.
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| CN114167059A (en) * | 2021-11-03 | 2022-03-11 | 郑州大学 | Biomarker for diagnosing esophageal squamous carcinoma and detection kit |
| CN116430049A (en) * | 2023-04-03 | 2023-07-14 | 汕头大学医学院 | Metabolic marker of esophagus cancer and application thereof |
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| CN116430049A (en) * | 2023-04-03 | 2023-07-14 | 汕头大学医学院 | Metabolic marker of esophagus cancer and application thereof |
| CN116430049B (en) * | 2023-04-03 | 2023-10-31 | 汕头大学医学院 | Metabolic markers of esophageal cancer and their applications |
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Application publication date: 20211029 |