CN117147737B - Plasma combined marker for esophageal squamous carcinoma diagnosis, kit and detection method - Google Patents
Plasma combined marker for esophageal squamous carcinoma diagnosis, kit and detection method Download PDFInfo
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- 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
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
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Abstract
The invention provides a plasma combined marker for esophageal squamous carcinoma diagnosis, a kit and a detection method, wherein the plasma combined marker comprises the following components: 3-methoxysalicylic acid, 2, 6-dihydroxybenzoic acid, hippuric acid, 4-hydroxymaleic acid and 2-hydroxyoctanoic acid; detecting the content of the combined marker in the blood plasma of the subject by using the kit, constructing a diagnosis model based on a binary logistic regression equation, calculating a combined marker variable, and judging whether the subject suffers from esophageal squamous cell carcinoma or not based on the determined cut-off value. The invention provides the plasma metabolic marker for auxiliary screening and diagnosis of esophageal squamous carcinoma for the first time, can accurately measure the content of the combined marker in the plasma, has 93.0 percent of specificity and 87.4 percent of sensitivity for diagnosis of esophageal squamous carcinoma, and performs diagnosis through an objective inspection index of the change of the content of endogenous micromolecular metabolites in the plasma of a patient, thereby simplifying the diagnosis process, obviously improving the diagnosis accuracy and having good clinical use and popularization values.
Description
Technical Field
The invention relates to the technical field of analytical chemistry and medical biological detection, in particular to a novel combined metabolic marker and application of a detection kit thereof in diagnosis of esophageal squamous cell carcinoma; in particular to a plasma combined marker for diagnosing esophageal squamous carcinoma, a kit and a detection method.
Background
Esophageal cancer (Esophageal cancer) is one of the most common malignant tumors worldwide, with the two major subtypes of esophageal cancer being squamous carcinoma (esophageal squamous cell carcinoma, ESCC) and adenocarcinoma (esophageal adenocarcinoma, EAC). The early stage esophageal squamous carcinoma patients have no obvious symptoms, most esophageal squamous carcinoma patients are in middle and late stages of the disease when diagnosis is confirmed, the prognosis is extremely poor, and the five-year survival rate is only 5% -15%, so that the early stage diagnosis of esophageal squamous carcinoma is very important.
The main means for diagnosing esophageal squamous carcinoma clinically at present comprise esophageal omentum cytological examination, X-ray barium meal radiography, esophageal ultrasonic endoscope, esophageal endoscopy and the like. However, these methods are invasive examination, complex in operation, low in diagnostic sensitivity and specificity, and expensive, so that development of a marker having clinical diagnostic potential and a detection method thereof are urgent.
Studies have shown that the occurrence of esophageal squamous carcinoma involves alterations in various metabolic pathways in humans, and is characterized by significant differences in the levels of various small molecule metabolites such as amino acids, fatty acids, lipids, etc., in plasma samples between esophageal squamous carcinoma patients and normal humans. Metabonomics is used as an emerging life histology means for searching disease biomarkers and characterizing biological metabolic pathways, and provides a brand-new view for researching the occurrence mechanism of diseases and finding disease markers. In recent years, successful cases of metabolomics in disease diagnosis have been reported in succession. For example, the content of sarcosine in urine was detected using liquid chromatography mass spectrometry to determine prostate cancer (Srekumar A, poisson L M, rajendiiran T M, et al Metabolomic profiles delineate potential role for sarcosine in prostate cancer progress. Nature, 2009, 457 (7231): 910-914.), and the combination markers glycocholic acid and phenylalanine-tryptophan were used for early liver cancer diagnosis and the like (Luo P, yin P, hua R, et al A large-scale, multi-center serum metabolite biomarkers identification study for the early detection of hepatocellular carcinoma. Hepatology, 2017, 67.).
Metabolic reprogramming is an important hallmark of tumors, esophageal squamous carcinoma is a malignant digestive tract tumor, which usually appears as a metabolic disorder of the whole body, so that metabonomics technology and methods are very suitable for researching esophageal squamous carcinoma. Currently, there have been studies on esophageal squamous cell carcinoma using metabonomics, for example, xu et al (Xu J, chen Y, zhang R, et al Global and targeted Metabolomics of esophageal squamous cell carcinoma discovers potential diagnostic and therapeutic biolackers Mol Cell Proteomics, 2013, 12 (5): 1306-18.), wang et al (Wang J, zhang T, shen X, et al Serum Metabolomics for early diagnosis of esophageal squamous cell carcinoma by UHPLC-QTOF/MS. Metabolic, 2016, 12 (7): 116), all of which have been studied using metabonomics.
However, most of the existing studies generally lack validation experiments, or the sample size is small, and the clinical reference value and credibility are low.
Disclosure of Invention
In view of the above, the present invention aims to solve the clinical problems of difficult diagnosis, high diagnosis cost, poor specificity, etc. caused by hidden symptoms of early stage esophageal squamous cell carcinoma, and to select a plasma combined marker by chemometric analysis from the discovery to the verification strategy, and to provide a kit and a detection method for detecting the combined marker, which are applicable to early stage esophageal squamous cell carcinoma diagnosis.
The present invention provides a plasma combination marker for diagnosis of esophageal squamous carcinoma, the plasma combination marker comprising: 3-methoxysalicylic acid, 2, 6-dihydroxybenzoic acid, hippuric acid, 4-hydroxymaleic acid and 2-hydroxyoctanoic acid.
The invention also provides a kit for diagnosis of esophageal squamous carcinoma formulated with reagents for detecting the level of a plasma combination marker as described above in a diagnostic sample of a subject, the kit comprising:
(1) Standard chemicals: the standard chemicals are respectively used for drawing quantitative standard curves of corresponding plasma metabolites;
(2) An extract containing stable isotope internal standard compounds: the extracting solution is used for preprocessing a plasma sample from a subject and is acetonitrile solution containing the following stable isotope internal standard compound, namely L-tryptophan-d 5;
(3) And (3) eluting the eluent used for eluting the chromatographic column.
Further, the eluent for eluting the chromatographic column is an eluent for eluting a ACQUITY UPLC HSS T3 (2.1x100 mm,1.8 μm, waters) chromatographic column, comprising: 0.1% (v/v) aqueous formic acid and 0.1% (v/v) acetonitrile formic acid.
Further, the subject is esophageal squamous carcinoma patient and healthy volunteer, and the diagnostic sample is subject plasma.
The invention also provides a plasma combined marker detection method for diagnosing esophageal squamous carcinoma, which uses the kit for diagnosing esophageal squamous carcinoma, and comprises the steps of calculating combined marker variables of a subject, wherein the calculation method of the combined marker variables comprises the following steps of:
(1) 3-methoxy salicylic acid, 2, 6-dihydroxybenzoic acid, hippuric acid, 4-hydroxy hippuric acid and 2-hydroxy octanoic acid are used as standard chemicals, an internal standard compound L-tryptophan-d 5 is added, and corresponding standard curves are respectively drawn;
(2) Pretreating a plasma sample from a subject with an extract, wherein the extract is an acetonitrile solution comprising an internal standard compound L-tryptophan-d 5; through 96-wellSirroco protein precipitation plates (Waters) filter proteins in plasma samples, and the filtrate was concentrated by centrifugation with water: acetonitrile=98:2 (v/v) redissolution, sample loading to chromatographic column through autosampler, eluting and separating, adopting mass spectrum to detect under negative ion mode, and ion pairs monitored by combined markers under triple quadrupole mass spectrum multiple reaction monitoring mode are respectively:m/z 167.1.fwdarw.108 (3-methoxysalicylic acid),m/z153- > 109 (2, 6-dihydroxybenzoic acid),m/z178.1- > 134 (hippuric acid),m/z194-100 (4-hydroxy hippuric acid),m/z159.1→113.1 (2-hydroxyoctanoic acid);
(3) Extracting ion flow graphs from each metabolite obtained based on liquid chromatography-mass spectrometry analysis, obtaining peak areas of each metabolite and an internal standard compound by adopting peak extraction software MultiQuant, and calculating the concentration of each metabolite in a plasma sample of a subject according to the quantitative standard curve of the step (1) and the eluting peak intensity of the step (2); calculating a combined marker variable based on a binary logistic regression analysis model to obtain diagnosis standards of esophageal squamous cell carcinoma; the joint equation of the binary logistic regression analysis model is: p=1/[ 1+e -(3.182-0.148×M1-0.003×M2-0.002×M3-0.259×M4-0.008×M5) ]Wherein M1 is the content of 2, 6-dihydroxybenzoic acid in blood plasma, M2 is the content of 2-hydroxyoctanoic acid, M3 is the content of hippuric acid, M4 is the content of 3-methoxysalicylic acid, and M5 is the content of 4-hydroxymaleic acid.
Based on the amount of marker combination in plasma, the combined marker variable P was calculated, and when the sum of sensitivity and specificity was maximum, the optimal cut-off value of the combined marker variable P was 0.6, indicating that when P was 0.6 or more, the sample could be from esophageal squamous carcinoma patients, whereas when P <0.6, the sample could be from healthy volunteers.
The technical scheme of the clinical practical application scene of the invention is as follows:
1) The metabonomics technology of ultra-high performance liquid chromatography-mass spectrometry is adopted to carry out metabolism profile analysis on plasma of healthy volunteers and esophageal squamous carcinoma patients, and metabolites are comprehensively screened through screening conditions such as p value, VIP value, change multiplying power, AUC value and the like, so that differential metabolites meeting the conditions are reserved;
2) Another batch of independent samples was used to verify the differential metabolites retained in step 1). Determining a combination marker which can be used for diagnosing esophageal squamous carcinoma;
3) Regression is carried out on the differential metabolites reserved in the step 2) into combined marker variables through a binary logistic regression analysis method, and the sensitivity and the specificity of the combined markers are evaluated by adopting ROC curves, and meanwhile, the combination with simplicity can be used as the combined markers;
4) Verifying the combined marker by using another group of plasma samples of healthy volunteers and esophageal squamous carcinoma patients, and determining that 3-methoxysalicylic acid, 2, 6-dihydroxybenzoic acid, hippuric acid, 4-hydroxy hippuric acid and 2-hydroxy octanoic acid can be used as the combined marker for assisting in diagnosing esophageal squamous carcinoma;
5) Use of combination markers: the in-vivo contents of 3-methoxysalicylic acid, 2, 6-dihydroxybenzoic acid, hippuric acid, 4-hydroxy hippuric acid and 2-hydroxy octanoic acid are regressed into a combined marker variable P by a binary logistic regression analysis method by using data statistical software SPSS, and the diagnosis standard of esophageal squamous carcinoma is obtained, wherein the combined equation is P=1/[ 1+e ] -(3.182-0.148×M1-0.003×M2-0.002×M3-0.259×M4-0.008×M5) ]Wherein M1 is the content of 2, 6-dihydroxybenzoic acid in blood plasma, M2 is the content of 2-hydroxyoctanoic acid, M3 is the content of hippuric acid, M4 is the content of 3-methoxysalicylic acid, and M5 is the content of 4-hydroxymaleic acid.
The resulting variable P increases in patients with esophageal squamous carcinoma, and the value of the variable can be used to assist in determining esophageal squamous carcinoma. Here, based on the sample involved in the experiment, the cut-off value (cut-off value) of the combined marker variable was set to 0.6, and if it is higher than the cut-off value, esophageal squamous cell carcinoma is likely to be the case, according to the principle of optimal diagnostic sensitivity and specificity, i.e., the principle of maximum area under ROC curve (AUC). A new equation can also be obtained through binary logistic regression according to the actual results of the experimenters, and the optimal intercept value of the laboratory is defined. Referring to FIG. 1A, an analysis of the ROC curve of the combination markers of the invention is shown; referring to FIG. 1B, the optimum cut-off values for the combination markers of the present invention are shown.
(6) A diagnostic system comprising: the chromatographic column was ACQUITY UPLC HSS T (2.1X100 mm,1.8 μm, waters) and the detection instrument was an ultra-high performance liquid chromatography tandem mass spectrometer using negative ion mode detection.
The invention adopts chromatographic mass spectrometry to analyze and detect the metabolites in the blood plasma, adopts a strategy from discovery to verification to screen out the combined markers through chemometric analysis, successfully screens out the combined markers which are based on human blood plasma and can be used for diagnosing esophageal squamous carcinoma, has good diagnosis sensitivity and specificity, and is expected to provide a new effective detection means for clinical diagnosis of esophageal squamous carcinoma.
Compared with the prior art, the invention has the beneficial effects that:
the plasma combined marker for esophageal squamous carcinoma diagnosis and the detection kit provide plasma metabolic markers for assisting in screening and diagnosing esophageal squamous carcinoma for the first time, have higher accuracy and diagnosis reference value, can accurately determine the content of the combined marker in plasma, and can be used for diagnosing esophageal squamous carcinoma with the specificity of 93.0% and the sensitivity of 87.4% by using the kit and a diagnosis prediction model thereof, so that diagnosis of esophageal squamous carcinoma is not completely dependent on invasive examination or imaging examination, but is carried out by using an objective examination index of the content change of endogenous micromolecular metabolites in plasma of a patient, the diagnosis process is simplified, the diagnosis accuracy is remarkably improved, and the kit has wide market development prospect.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention.
In the drawings:
FIG. 1A is a graph of ROC curve analysis of a combination marker according to an embodiment of the present invention;
FIG. 1B is a graph of the optimum cut-off values of the combined markers according to the embodiment of the invention.
Detailed Description
Reference will now be made in detail to exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, the same numbers in different drawings refer to the same or similar elements, unless otherwise indicated. The implementations described in the following exemplary examples are not representative of all implementations consistent with the present disclosure. Rather, they are merely examples of apparatus and products consistent with aspects of the present disclosure.
The terminology used in the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used in this disclosure, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any or all possible combinations of one or more of the associated listed items.
It should be understood that although the terms first, second, third, etc. may be used in this disclosure to describe various information, these information should not be limited to these terms. These terms are only used to distinguish one type of information from another. For example, first information may also be referred to as second information, and similarly, second information may also be referred to as first information, without departing from the scope of the present disclosure. The word "if" as used herein may be interpreted as "at … …" or "at … …" or "responsive to a determination", depending on the context.
Embodiments of the present invention are described in further detail below.
Example 1
Quantitative analysis of esophageal squamous carcinoma diagnosis plasma combination markers:
1. plasma sample collection:
the plasma sample collection included 500 esophageal squamous carcinoma patients and 500 healthy volunteers, all of whom were confirmed to have cancer by clinical histopathology, and all of which were healthy persons with age matched patients who had not had cancer. All were blood collected the next morning after 8 hours of fasting. Blood samples collected in EDTA-K 2 Standing in an anticoagulation tube, centrifuging at 4000 rpm/min for 10 minutes,the plasma was taken and stored in a-80 ℃ refrigerator for further use.
2. The analysis method comprises the following steps:
2.1 pretreatment of plasma samples:
thawing the blood plasma at 4 ℃, taking 100 mu L of blood plasma sample, and adding 300 mu L of extracting solution to precipitate protein; proteins in plasma samples were filtered through 96-well Sirroco protein precipitation plates (Waters, USA) and the filtrate was concentrated by centrifugation and stored in a-80℃freezer. Before sample injection, the samples were reconstituted with 100 μl of water, acetonitrile=98:2 (v/v), and after shaking, 10000 rpm/min,4 ℃, centrifuged for 5 minutes, and 90 μl of supernatant was taken for subsequent analysis.
2.2 Ultra-high performance liquid chromatography mass spectrometry:
(1) Chromatographic conditions: waters ACQUITY UPLC system (Waters company, usa); chromatographic column: ACQUITY UPLC HSS T3 column (2.1X100 mm,1.8 μm, waters); mobile phase A is 0.1% (v/v) formic acid aqueous solution, mobile phase B is 0.1% (v/v) formic acid acetonitrile solution; elution gradient: 0-5 min is 2% B phase to 30% B phase, 5-11 min is 30% B phase to 100% B phase, and holding for 4 min; column temperature of 35 o C, performing operation; the flow rate of the mobile phase is 0.25 mL/min; the sample loading was 5. Mu.L.
(2) Mass spectrometry conditions:
quadrupole-linear ion trap tandem mass spectrometer (QTRAP) TM 5500, applied Biosystems/MDS SCIEX company) using a multi-reaction monitoring mode (scheduled-MRM) with a specific acquisition time window to scan and detect metabolites, anion voltage of-4500V, ion source temperature of 500 o C, the shielding Gas (Curtain Gas) was set at 35 psi, the first assist Gas (Gas 1) was set at 60 psi, and the second assist Gas (Gas 2) was set at 50 psi. Ion pairs monitored by the marker in a triple quadrupole mass spectrometry multiple reaction monitoring mode are respectively as follows:m/z 167.1.fwdarw.108 (3-methoxysalicylic acid),m/z153- > 109 (2, 6-dihydroxybenzoic acid),m/z178.1- > 134 (hippuric acid),m/z194-100 (4-hydroxy hippuric acid),m/z159.1.fwdarw.113.1 (2-hydroxyoctanoic acid).
2.3 plasma test results and auxiliary diagnostic methods:
the method is matched with the use of a kit to quantitatively analyze the contents of 3-methoxysalicylic acid, 2, 6-dihydroxybenzoic acid, hippuric acid, 4-hydroxy hippuric acid and 2-hydroxy octanoic acid in plasma samples of esophageal squamous carcinoma patients and healthy volunteers, and the combined marker variable P is calculated by using model binary logistic regression analysis, so that esophageal squamous carcinoma patients and healthy volunteers can be well distinguished.
In this example, the area under the ROC curve (AUC value) was 0.966, and the sensitivity and specificity were 87.4% and 93.0%, respectively.
Example 2
Multicenter verification of esophageal squamous carcinoma diagnosis plasma combination markers:
1. plasma sample collection:
plasma samples were taken from 155 esophageal squamous carcinoma patients (ESCC group), 171 healthy volunteers (HC group), wherein plasma samples from esophageal squamous carcinoma patients were provided by 3 different hospitals (Center 1, center2, center 3), respectively, and healthy human plasma age-matched with esophageal squamous carcinoma patients was provided by volunteers. The preparation process of the sample comprises collecting fasting venous blood 2 mL, placing in EDTA-K 2 Anticoagulation vacuum blood collection tube, mixing, centrifuging at 3000 rpm for 10 min, collecting supernatant, and freezing in-80 o And C, a refrigerator.
2. The analysis method comprises the following steps:
2.1 pretreatment of plasma samples:
as in example 1.
2.2 Ultra-high performance liquid chromatography mass spectrometry:
as in example 1.
2.3 plasma test results and auxiliary diagnostic methods:
by the method of example 2, 2.1 and the use of the detection kit, the contents of 3-methoxysalicylic acid, 2, 6-dihydroxybenzoic acid, hippuric acid, 4-hydroxyhippuric acid and 2-hydroxyoctanoic acid in plasma samples of esophageal squamous carcinoma patients and healthy volunteers are quantitatively analyzed, unknown samples can be rapidly judged according to the calculated combined marker variable P by taking 0.6 as a cut-off value, the judging result is compared with the actual grouping of the samples, the result is shown in the table 1, the judging accuracy of the combined marker variable P on the samples of different centers is higher than 70%, and the false positive rate and the false negative rate are 18.13% and 19.35%, respectively. The result shows that the diagnosis model constructed by the combined marker has high sensitivity and higher clinical application value.
TABLE 1 Multi-center validation results for combination markers
The use of the combination marker and the esophageal squamous carcinoma detection kit described above with reference to examples are illustrative and not limiting, and several examples can be listed according to the scope of the invention. It will be appreciated by those skilled in the art that equivalent modifications or substitutions for the relevant technical features can be made by those skilled in the art without departing from the principles of the invention, and those modifications or substitutions will be within the scope of the invention.
The foregoing description is only of the preferred embodiments of the invention and is not intended to limit the invention; various modifications and variations of the present invention will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. Plasma composition marker for diagnosis of esophageal squamous carcinoma, characterized in that it consists of 3-methoxysalicylic acid, 2, 6-dihydroxybenzoic acid, hippuric acid, 4-hydroxymaleic acid and 2-hydroxyoctanoic acid.
2. Use of a plasma composition marker according to claim 1 for the preparation of a kit for diagnosis of esophageal squamous carcinoma, said kit comprising:
(1) Standard chemicals: the standard chemicals are respectively used for drawing quantitative standard curves of corresponding plasma metabolites;
(2) An extract containing stable isotope internal standard compounds: the extracting solution is used for preprocessing a plasma sample from a subject, and is acetonitrile solution containing a stable isotope internal standard compound, wherein the stable isotope internal standard compound is L-tryptophan-d 5;
(3) And (3) eluting the eluent used for eluting the chromatographic column.
3. Use of a plasma composition marker according to claim 2 in the preparation of a kit for diagnosis of esophageal squamous carcinoma, wherein the eluent for eluting a chromatographic column is for eluting ACQUITY UPLC HSS T3: 2.1X100 mm,1.8 μm, eluent from a Waters column comprising: 0.1% (v/v) aqueous formic acid and 0.1% (v/v) acetonitrile formic acid.
4. Use of a plasma composition marker according to claim 3 for the preparation of a kit for diagnosis of esophageal squamous carcinoma, wherein the subject is esophageal squamous carcinoma patient and healthy volunteers and the diagnostic sample is the subject's plasma.
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| Title |
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| Combined metabolomic analysis of plasma and tissue reveals a prognostic risk score system and metabolic dysregulation in esophageal squamous cell carcinoma;Zhongjian Chen 等;Frontiers in Oncology;第10卷;第1-13页 * |
| 基于液相色谱—质谱联用技术的哈萨克族食管鳞状细胞癌代谢组学研究;张海平;中国博士学位论文全文数据库(第3期);E072-41 * |
| 血清肿瘤标志物Cyfra 21-1、SCCAg、Ferritin、CEA、CA19-9和AFP对口腔/口咽鳞癌的诊断价值;袁川淑 等;重庆医科大学学报;第42卷(第9期);第1066-1071页 * |
| 食管癌非创伤性检查的现状与进展;林金欢 等;国际消化病杂志;第35卷(第4期);第258-262页 * |
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