A kind of blood serum metabolic group Epidemiological Analysis modelTechnical field
The present invention relates to a kind of blood serum metabolic group Epidemiological Analysis model, convenient, fastly can carry out early stage preliminary examination to the cancer of the esophagus by this model, belong to analysis technical field.
Background technology
The cancer of the esophagus (esophageal cancer) is the malignant change formed by esophagus squameous epithelium or epithelioglandular paraplasm, is by the process that gradually develop of atypical hyperplasia to cancer.Show according to World Health Organization's latest data: the whole world about has 400,000 people to die from the cancer of the esophagus every year, and China is the country that Incidence of esophageal cancer and mortality ratio are the highest, and the organization type of 90% patient is squamous cell carcinoma.River in Shangdong Province, Shandong Province river valley is Esophageal Cancer in High Risk Areas, the highest with Feicheng, Ningyang County and Wenshang County, the incidence of disease is respectively 93.95/10 ten thousand, 88.68/10 ten thousand and 62.26/10 ten thousand, far above national mean level (16.7/10 ten thousand), especially Feicheng, its incidence of disease is 3.7 times of Shandong Province's average level, accounts for 50% of whole county Death Causes of Tumor.The high incidence of the cancer of the esophagus and high mortality cause huge Disease Spectrum and financial burden to China especially river in Shangdong Province, Shandong Province river valley, have become serious public health problem, urgently to be resolved hurrily.
The cause of disease of the cancer of the esophagus is complicated, most scholar thinks gene and the coefficient result of environment, the region aggregation of Incidence of Esophageal Cancer points out it relevant to environmental factor and bad life habits, as vitamin in dietary ingredient and microelement deficiencies, take food containing the more food of nitrosamines, as like pickling sauerkraut or the food that goes mouldy, for a long time like eat into scalding, smoking, bad habit etc. of drinking.That what molecules research showed the cancer of the esophagus is is multifactor, the multistage, the accumulation of polygenic variation and each factor and the interactional result of environmental factor, relate to the change of numerous proto-oncogene, tumor suppressor gene and protein, and the cell cycle.Very long process is often experienced in generation from precancerous lesion to the cancer of the esophagus, the important precancerous lesion of the cancer of the esophagus be esophagus squameous epithelium light, in, severe atypical hyperplasia, generally need several years even more than ten years by slight atypical hyperplasia to canceration.If during this period, precancerous lesion crowd changes bad life habits, carries out preventative taking medicine, and so will greatly reduce the probability of its canceration.Therefore, the EARLY RECOGNITION of precancerous lesion of cancer of esophagus seems particularly important with intervening to its canceration rate of reduction.
For examination be in slightly, the precancerous lesion state of moderate and severe atypical hyperplasia or the case of the early stage cancer of the esophagus, country from 2008 Feicheng, Shandong Province (the national cancer of the esophagus is early examined and is early controlled Demonstration Base), for the crowd of all 40 ~ 69 years old in Demonstration Base, by the indicative Biopsy of iodine staining under gastroscope, carry out the cancer of the esophagus and early examined and early control project.Carrying out histopathological examination to suspicious Mucosa Biopsy after the inspection of oesophagus scope iodine staining, is the goldstandard method in the generaI investigation of current Esophageal Cancer in High Risk Areas, people at highest risk's screening and early diagnosis.Because it is directly perceived, Tu Xiang Cheongju is clear and specificity is high, does not also have a kind of method to substitute its status at present.Although the method accuracy is high, but still there is certain limitation: gastroscope is for there being wound inspection, and person under inspection feels painful, has in the process of implementation and does not comply with phenomenon; Iodine staining indicative Biopsy complicated operation under gastroscope, cost are higher, if the feasibility promoted outside demonstration area occurred frequently and efficiency still remain to be discussed; The iodine staining positive rate of Esophageal Cancer in High Risk Areas is less than 10%, and the examination crowd that treats up to 90% will bear the gastroscopic misery of wound, returns country and early examines early that controlling project brings unnecessary financial burden.Therefore, be badly in need of a kind of easier method newly of exploitation, seek the change at cancer of the esophagus generation developing stage organismic internal environment, goldstandard screening methods (i.e. the indicative Biopsy of iodine staining under gastroscope) for the cancer of the esophagus provides scientific basis and the translational medicine support of primary dcreening operation, to distinguish iodine staining feminine gender and iodine staining Positive Populations, thus avoid iodine staining Population with Negative to bear the painful of wound gastroscope and reduce morning examining the expenditure early controlling project.In addition, early examine in the cancer of the esophagus and early control that project is once tentative in early days uses the preliminary examination cancer of the esophagus high-risk individuals of Harvard's risk of cancer forecast model (content that questionnaire relates to comprises five broad aspect such as living environment, diet style and custom, mood and mood, medical history and Family history of cancer), then implement the indicative Biopsy of iodine staining under gastroscope again for high-risk individuals.But in reality performs, find that it has higher false negative rate (namely failing to pinpoint a disease in diagnosis), ROC area under curve AUC is 0.70 (95%CI, 0.66 – 0.74) (literature reference: Thrift AP, Kendall BJ, Pandeya N, Vaughan TL, Whiteman DC. A clinical risk prediction model for Barrett esophagus. Cancer Prev Res (Phila), 2012,5 (9): 1115-23.).Because this traditional prescreening method effect is undesirable, current cancer of the esophagus examination scope only do 40-69 year age limit.
Metabolism group carries out qualitative and quantitative detection to all molecular weight in biological sample (as serum, urine, saliva etc.) lower than 1000Da small molecule metabolites (as biological micromolecules such as fatty acid, amino acid, nucleosides and steroidals), thus the metabolism that monitoring body is made by endogenous material after the interference such as disease or hazards accumulation responds.Biological information in body passes to protein by gene through transcribing, and is finally presented as small molecule metabolites.Be different from the biosome inherent difference of genomics and protein science reflection, the research field of metabolism group extend to influencing each other and acting between body and environment.Small molecule metabolites is not only the material base of body vital movement, biochemical metabolism, also embody the change of some foeign element to internal metabolism environment, thus some unique metabolic thing concentration interindividual difference in fact reflect in disease performance and the external cause of disease.Recent study finds, in the disease development processes such as such as metabolic disease and malignant tumour (oophoroma), the metabolism of body basic biochemistry all there occurs significant change, to play a significant role to the metabolic mechanism of human intelligible complex disease, simultaneously for the screening of complex disease and early diagnosis provide brand-new technical method.
The cancer of the esophagus is a kind of typical metabolic disease, and be esophageal squamous cell carcinoma (ESCC) China more than 90% patient, it is usually expressed as the metabolic disorder of animal economy, and therefore metabonomic technology and method are very suitable for the research of the cancer of the esophagus.At present, someone utilizes metabolism group to study the cancer of the esophagus, such as (the Wu H such as Wu, Xue R, Lu C, et al. Metabolomic study for diagnostic model of oesophageal cancer using gas chromatography/mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci, 2009, 877 (27): 3111-7.), (the Zhang J such as Zhang, Bowers J, Liu L, et al. Esophageal cancer metabolite biomarkers detected by LC-MS and NMR methods. PLoS One, 2012, 7 (1): e30181.), (the Xu J such as Xu, Chen Y, Zhang R, et al. Global and targeted metabolomics of esophageal squamous cell carcinoma discovers potential diagnostic and therapeutic biomarkers. Mol Cell Proteomics, 2013, 12 (5): 1306-18.) metabonomic technology is all utilized to be studied the cancer of the esophagus, but these researchs concentrate on pathology and pathogenetic research mostly on the whole, genesis mechanism for disease provides new clue, the sample used also is the sample of the advanced esophageal carcinoma patient based on hospital's collection, the result of study of gained only can find the metabolic profile difference that Incidence of Esophageal Cancer is compared with normal healthy controls late period, and the blood serum metabolic situation of cancer of late stage and the blood serum metabolic difference of precancerous lesion huge, can not help for the early stage screening of the cancer of the esophagus and early diagnosis band.Further, current above-mentioned research method is easily subject to the impact of selectivity bias on normal healthy controls is chosen, and causes the generalization of evaluation result poor.
Summary of the invention
Loaded down with trivial details for cancer of the esophagus methods for screening in prior art, costly, to deficiencies such as detection crowd cause suffering, also there is no a kind of simple and efficient, applicable large-scale defect, the invention provides a kind of blood serum metabolic group Epidemiological Analysis model, this analytical model construction method is simple, under can replacing gastroscope, the indicative Biopsy of iodine staining carries out early stage, the preliminary examination of the cancer of the esophagus to crowd, both economical and practical, convenient and swift, the misery of crowd to be detected can be avoided again, easy to utilize.
The present invention is directed to the deficiency adopting at present the indicative Biopsy of iodine staining under gastroscope the cancer of the esophagus to be carried out to preliminary examination, propose to adopt blood serum metabolic omics technology to replace the indicative Biopsy of iodine staining under gastroscope the cancer of the esophagus to be carried out to the thinking of preliminary examination first.The cancer of the esophagus screening that the present invention relies on " the national cancer of the esophagus is early examined and early controlled Demonstration Base (Feicheng, Shandong Province) " with follow up a case by regular visits to crowd's queue, for the indicative biopsy object of iodine staining under gastroscopes all in project, gather the first Lesions in Upper Gastrointestinal Tract found, the serum specimen of precancerous lesion of cancer of esophagus and cancer of the esophagus early stage patient, and to randomly draw in screening without the health objects of Lesions in Upper Gastrointestinal Tract as a control group, use the high flux of quick separating liquid chromatography and mass spectrometer (LC/MS) to detect and obtain corresponding Metabolic fingerprinting, and by obtaining model to the further analysis structure of collection of illustrative plates, this model can be analyzed and distinguish serum protein moteblites situation, tentatively can judge whether that there is the ill danger of the cancer of the esophagus by serum protein moteblites situation.Utilize model of the present invention to may be used for the early stage screening of total man group's cancer of the esophagus of high region of disease, screen out the crowd not having ill danger, and then carry out conventional endoscopic inspection for high-risk individuals, judge whether to suffer from the cancer of the esophagus.The present invention reduces Endoscopic scope, has important economic and social benefit for the enforcement of cancer of the esophagus screening and popularization.
The concrete technical scheme of the present invention is as follows:
A kind of blood serum metabolic group Epidemiological Analysis model, construction method comprises the following steps:
(1) healthy serum sample and ill serum sample is collected, as analyzing samples;
(2) adopted by each analyzing samples LC-MS blood serum metabolic omics technology to analyze, obtain the original Metabolic fingerprinting of healthy serum sample and ill serum sample;
(3) the original Metabolic fingerprinting of healthy serum sample and ill serum sample is carried out collection of illustrative plates pre-service, obtain every behavioural analysis sample, be often classified as the two-dimensional matrix of metabolin information, for further statistical study;
(4) two-dimensional matrix of step (3) is carried out principal component analysis (PCA) (PCA) and partial least squares discriminant analysis (PLS-DA) successively, obtain PLS-DA model, verify the PLS-DA model obtained, dangerous without over-fitting, then gained PLS-DA model is blood serum metabolic group Epidemiological Analysis model.
In above-mentioned construction method, described LC-MS blood serum metabolic omics technology is the method for the Metabolic fingerprinting adopting liquid chromatograph mass spectrography systems axiol-ogy serum, in the preferred embodiment of the present invention, liquid chromatograph mass spectrography system used is Ultra Performance Liquid Chromatography-high resolution mass spectrum combined system (UPLC-QTOF/MS).Preferably, liquid chromatography chromatographic column used is Waters ACQUITY UPLChSS T3(1.8 μm; 100 mm (length) × 2.1 mm) chromatographic column, sample size is 6 μ L, and injector temperature is 4 DEG C, and flow velocity is 0.5 ml/min.It is 0.1wt% aqueous formic acid (positive ion ESI+) or 0.5mmol/L ammonium fluoride aqueous solution (negative ion ESI-) that chromatogram flow phase comprises two kinds of solvent: A, and B is acetonitrile solution (positive ion ESI+) or the pure acetonitrile (negative ion ESI-) of 0.1wt% formic acid.Chromatogram condition of gradient elution is: 0-1min is 1%B, 1-8min is that 1%B-100%B increases progressively gradually, and then 10-10.1min is that 100%B is kept to 1%B rapidly, and then 1%B continues 1.9min.Preferably, Mass Spectrometer Method uses quadrupole rod time-of-flight mass spectrometry instrument Q-TOF, and adopts positive ion mode (ESI+) and the negative ion mode (ESI-) of electric spray ion source.Ion source temperature is set as 400 DEG C, and taper hole airshed is 12L/min.Meanwhile, desolventizing air temperature set is 250 DEG C, desolventizing airshed 16L/min.Under positive ion and negative ion mode, capillary voltage is respectively+3kV and-3kV, and taper hole voltage is 0V.Taper hole pressure is 20psi(positive ion) and 40psi(negative ion).The mass charge ratio range of spectrum data collection is 50 ~ 1200 m/z, and the sweep frequency of collection is 0.25s.
In above-mentioned construction method, described ill serum sample is selected from the serum of esophagitis patient, slight atypical hyperplasia patient, severe atypical hyperplasia patient, moderate atypical hyperplasia patient, precancerous lesion of cancer of esophagus patient, cancer of the esophagus early stage patient and carcinoma in situ patient.Described patients serum's sample can be the serum of the patient suffering from above-mentioned a kind of illness, also can be suffer from two or more the serum of patient of illness above-mentioned (such as ill serum is the serum of esophagitis patients serum and slight atypical hyperplasia patient, or severe atypical hyperplasia patients serum, moderate atypical hyperplasia patients serum and cancer of the esophagus early stage patient serum), preferably, the serum that ill serum sample contains the patient suffering from above-mentioned often kind of illness (namely contains esophagitis in ill serum sample simultaneously, slight atypical hyperplasia, severe atypical hyperplasia, moderate atypical hyperplasia, precancerous lesion of cancer of esophagus, the serum of the early stage and carcinoma in situ patient of the cancer of the esophagus), prediction accuracy is higher.
In above-mentioned construction method, described healthy serum sample for there is no the serum of the crowd of disease or the disease similar with corresponding disease corresponding to ill serum sample, the serum of to be alternatively healthy serum sample the be crowd not having Lesions in Upper Gastrointestinal Tract.
In above-mentioned construction method, the R2X=0.231 of gained PLS-DA model, R2Y=0.749, Q2cum=0.638.Model sensitivity is high in the case, and specificity is good, has good extrapolation effect.
In above-mentioned construction method, described healthy serum sample and ill serum sample are all from 40-69 year crowd.
In above-mentioned construction method, selected serum sample quantity is large, and described ill serum sample 453, healthy serum sample 187, prediction effect is higher.
In above-mentioned construction method, in order to quality control situation when timely monitor model builds, add Quality control samples and carry out quality control.Feed postition is: every 10 analyzing samples add a Quality control samples, and this Quality control samples may be used for Real Time Monitoring sample from sample introduction pre-treatment to the quality control situation analytic process.Described Quality control samples composition is identical, is the sample that same healthy serum sample and same ill serum sample are mixed to get according to the volume ratio of 1:1.
In above-mentioned construction method, after obtaining analyzing samples and Quality control samples, pre-service to be carried out to them, add liquid chromatograph mass spectrography system after pre-service again and detect.Pre-service before sample introduction comprises the following steps:
(1) extract 50 μ l analyzing samples or Quality control samples with pipettor, be placed on 96 orifice plates of the automatic sample processing system (Agilent, USA) of Bravo;
(2) add 150 μ l methyl alcohol to extract, vortex 30s, and hatch with protein precipitation at-20 DEG C.
(3) then in supercentrifuge at 4 DEG C with 4000 revs/min of centrifugal 20min;
(4) supernatant of step (3) is poured in LC-MS sample injection bottle, detect in order to LC-MS at being kept at-80 DEG C.
The automatic sample processing system of above-mentioned Bravo, is also Bravo automatic fluid processing platform.
In above-mentioned analyzing samples and Quality control samples preprocessing process, if analyzing samples and Quality control samples longer for sample time, they can be put into-80 DEG C of refrigerators to preserve, during pre-service, the 0.5ml centrifuge tube that freezing 0.4ml analyzing samples or Quality control samples are housed is placed in water, surface level was advisable not have frozen surfaces, put into cold compartment of refrigerator and thawed; After thawing completely, vortex 30s.Pre-service is carried out by above-mentioned pre-treatment step again after process of thawing above.
In above-mentioned construction method, carry out collection of illustrative plates pre-service to original Metabolic fingerprinting to refer to: with Masshunter software, the original Metabolic fingerprinting obtained is converted to MZdata data file, then XCMS software package Mzdata data file is used to carry out comprising the pretreatment operation of aliging in retention time correction, peak identification, peak match and peak, obtain the two-dimensional matrix that can be used for statistical study, every behavioural analysis sample in matrix or Quality control samples, be often classified as metabolin information.
In above-mentioned blood serum metabolic group Epidemiological Analysis model, the metabolin information of healthy serum sample and ill serum sample lays respectively at the diverse location of model, and respective zero lap, can be designated as healthy serum sample group region is negative areas, and ill serum sample group region is positive region.
Use above-mentioned blood serum metabolic group Epidemiological Analysis model, may be used for the metabonomic analysis of serum sample.
The method of serum analysis metabolism group of the present invention, comprises the following steps: serum sample to be checked is carried out pre-service, reaches sample introduction requirement; LC-MS blood serum metabolic omics technology is adopted by pretreated serum sample to be checked to analyze, must the original Metabolic fingerprinting of this serum sample to be checked; This original Metabolic fingerprinting is carried out collection of illustrative plates pre-service, obtains the metabolin information that may be used for statistical study; This metabolin information is imported in blood serum metabolic group Epidemiological Analysis model, for analyzing the metabolism group situation of serum sample to be checked.By analyzing the prediction probability (be greater than 50% and be the positive) that can obtain cancer of the esophagus early screening risk, thus judge cancer of the esophagus examination risk by body metabolism situation.In actual applications, if the metabolin information of serum sample to be checked is positioned at ill serum sample group region (the i.e. positive region of model, namely prediction probability is greater than 50%), then represent that this people has the ill danger of the cancer of the esophagus, indicative biopsy under needing to carry out further scope; If the metabolin information of serum sample to be checked is positioned at the healthy serum sample group region (i.e. negative areas, prediction probability is less than 50%) of model, then represents that this people does not have the ill danger of the cancer of the esophagus, do not need further to check.
In the method for above-mentioned serum analysis metabolism group, pretreated step is carried out to serum sample to be checked, the original Metabolic fingerprinting of serum sample to be checked to be carried out the pretreated step of collection of illustrative plates all identical with pre-treatment step during above-mentioned structure blood serum metabolic group Epidemiological Analysis model, collection of illustrative plates pre-treatment step.
Screening Platform that the present invention relies on " the national cancer of the esophagus is early examined and early controlled Demonstration Base (Feicheng) ", gather esophagitis, atypical hyperplasia (being again atypical hyperplasia, dysplasia), precancerous lesion and the serum specimen in early stage cancer of the esophagus stage, selected serum specimen is up to 640 examples, scope is wide, quantity is many, and authenticity is more reliable.Obtain serum protein moteblites analytical model by UPLC-QTOF/MS and statistical pattern recognition method, this model as the model of the early stage screening of Esophageal Cancer area crowd's cancer of the esophagus, can reach the object of the early screening to the cancer of the esophagus.
Early stage or precancerous serum sample obtains based on the cancer of the esophagus for the method for blood serum metabolic group Epidemiological Analysis model of the present invention and serum analysis metabolism group, compared to the model that the serum sample of the cancer of the esophagus middle and advanced stage patient based on hospital obtains, blood serum metabolic group situation of the present invention with want the blood serum metabolic group situation matching degree of the people at highest risk of examination higher, be more suitable for the early screening of the cancer of the esophagus.And by the optimization to modeling method, model sensitivity of the present invention is high, and specificity is good, well can differentiate healthy population and high-risk patient groups, be very applicable to clinical practice.
Blood serum metabolic group Epidemiological Analysis technology is used in cancer of the esophagus early screening by the present invention first, preliminary examination can be carried out to the total man group of Esophageal Cancer in High Risk Areas by the present invention, filter out Esophageal Cancer crowd fast, easily, accuracy is high, reduces the scope of cancer of the esophagus examination.Cancer of the esophagus screening method becomes first through the method examination of serum analysis metabolism group of the present invention from iodine staining indicative Biopsy in Screening method under traditional direct gastroscope by the present invention, screening results be positive individuality again under row gastroscope the indicative Biopsy of iodine staining carry out examination, only need during serum analysis metabolism group to gather serum, without wound, spend low, effectively improve the efficiency of the full Mass screening in Esophageal Cancer in High Risk Areas, greatly reduce examination cost, effectively prevent the misery that part population has wound gastroscope to cause, there is important economic and social benefit, easy to utilize.In addition, the proposition of the inventive method also helps the easy doubtful sufferer of the discovery cancer of the esophagus, and the morning being conducive to cancer finds, early treatment, has very high scientific research and medical value.
Accompanying drawing explanation
Fig. 1 is the quality control chart that blood serum metabolic group detects, and transverse axis is retention time, and the longitudinal axis is the RSD% value of metabolin in QC sample.
Fig. 2 is the PCA shot chart of metabolic profile preanalysis, and wherein NEG is negative, represents healthy serum sample, and POS is positive, and represent ill serum sample, QC represents Quality control samples.
Fig. 3 is the three-dimensional shot chart of PLS-DA, the R2X=0.231 of modeling, R2Y=0.749, Q2cum=0.638, and wherein Screening NEG is that examination is negative, represents healthy serum sample, and Screening POS is that examination is positive, represents ill serum sample.
Fig. 4 is the PLS-DA modeling proof diagram based on random permutation method.
Fig. 5 is the external certificate shot chart of the PLS-DA model for cancer of the esophagus early screening, wherein Screening NEG is that examination is negative, represent healthy serum sample, Screening POS is that examination is positive, represent ill serum sample, Screening NEG-test represents that the examination of external testing sample is negative, and Screening POS-test represents that the examination of external testing sample is positive.
Fig. 6 is the ROC curve of PLS-DA model external testing sample.
Embodiment
Below, by following embodiment, the present invention is further explained, and advantage of the present invention is further proved.
construction method and the compliance test result of serum protein moteblites analytical model of the present invention are as follows:
1, research object
The cancer of the esophagus examination that this research relies on " the national cancer of the esophagus is early examined and early controlled Demonstration Base (Feicheng, Shandong Province) " with follow up a case by regular visits to community-based population queue, for Feicheng, Shandong Province 40-69 year gastroscope under the indicative biopsy object of iodine staining (confirming as goldstandard), gather and first to find the serum sample of the positive person under inspection of iodine staining (comprising esophagitis, slight atypical hyperplasia, severe atypical hyperplasia, moderate atypical hyperplasia, precancerous lesion of cancer of esophagus, cancer of the esophagus early stage patient, carcinoma in situ patient) of (without treatment or do not take medicine) as ill sample;and to randomly draw in screening iodine staining negative subject under gastroscope namely without the health objects of Lesions in Upper Gastrointestinal Tract as healthy sample.
This research detects 640 people altogether, wherein the positive person under inspection's (i.e. ill sample) of iodine staining totally 453 examples, the negative person under inspection's (i.e. healthy sample) of iodine staining totally 187 examples; Age, sex do not have statistical significance at two group differences, have comparability.
2, the blood serum metabolic group of LC-MS detects
Gather the serum of clinical samples and healthy sample respectively, as patients serum's sample and healthy serum sample, be put in after the serum sample of all collections is centrifugal in-80 DEG C of refrigerators and preserve, use Ultra Performance Liquid Chromatography-GC-MS (UPLC-QTOF/MS 6550, and the automatic Pretreated system of Bravo (Agilent Agilent), USA) metabolism group detection (point 3 large batch detection is carried out, and carry out quality control), obtain the original Metabolic fingerprinting comprising chromatogram and Information in Mass Spectra of sample.Concrete operations are as follows:
2.1 instrument and equipment
Experimental facilities comprises: UPLC-QTOF/MS 6550 system (Agilent, USA), Bravo system (Agilent, USA), high speed low temperature centrifugal machine, vibration scroll machine, nitrogen drying device, 4 DEG C of cold storage refrigerators (Haier), pure water instrument (Siemens).
Experiment consumptive material comprises: Waters ACQUITY UPLChSS T3 (particle size, 1.8 μm; 100 mm (length) × 2.1 mm) chromatographic column, liquid nitrogen, High Purity Nitrogen; Cone end sample injection bottle, 2ml centrifugal rotor, 2ml centrifuge tube (round bottom), pipettor, 1000 μ l rifle heads, 200 μ l rifle heads, marking pen, emgloves, mouth mask.
Experiment reagent comprises: methyl alcohol (enlightening horse, HPLC level is pure), acetonitrile (enlightening horse, HPLC level is pure), formic acid (recovery precision chemical research institute, Tianjin), pure water (TOC<10ppb).
2.2 serum sample pre-service
Before serum sample pre-service, prepare 60 parts of Quality control samples (QC), all patients serum's samples, healthy serum sample and Quality control samples are carried out random number, using patients serum's sample and healthy serum sample as analyzing samples, add a Quality control samples every 10 analyzing samples.Quality control samples is mixed same patients serum's sample and same healthy serum sample, and be divided into 60 parts equably.Patients serum's sample, healthy serum sample and Quality control samples all carry out pre-service, and pre-service comprises following 4 steps:
(1) extract 50 μ l analyzing samples or Quality control samples with pipettor, be placed on 96 orifice plates of the automatic sample processing system (Agilent, USA) of Bravo;
(2) add 150 μ l methyl alcohol to extract, vortex 30s, and hatch with protein precipitation at-20 DEG C.
(3) then in supercentrifuge at 4 DEG C with 4000 revs/min of centrifugal 20min;
(4) supernatant of step (3) is poured in LC-MS sample injection bottle, detect in order to LC-MS at being kept at-80 DEG C;
2.3 serum UPLC-QTOF/MS detect
The pretreated sample of 6 μ L equal portions is injected ACQUITY UPLC HSS T3 (particle size, 1.8 μm by UPLC system (1290 series, Agilent); 100 mm (length) × 2.1 mm) chromatographic column (Waters, Milford, USA).Loading sequence is completely random sample introduction, to get rid of the bias that Loading sequence brings.It is that (water dilutes 0.1wt% formic acid that chromatogram flow phase comprises two kinds of solvent: A, positive ion ESI+) or (the water dilution of 0.5mM ammonium fluoride, negative ion ESI-), B is 0.1wt% formic acid (dilution in acetonitrile, positive ion ESI+) or 100% acetonitrile (negative ion ESI-).Chromatogram gradient is: 0-1min is 1%B, 1-8min is that 1%B-100%B increases progressively gradually, and then 10-10.1min is that 100%B is kept to 1%B rapidly, and then 1%B continues 1.9min.Flow velocity is 0.5 ml/min.Whole sample detection process maintains 4 DEG C.Wherein, the percentage composition of A and B refers to volumn concentration.
Mass Spectrometer Method uses Agilent quadrupole rod time-of-flight mass spectrometry instrument Q-TOF (6550, Agilent), and adopts positive ion mode (ESI+) and the negative ion mode (ESI-) of electric spray ion source.Ion source temperature is set as 400 DEG C, and taper hole airshed is 12L/min.Meanwhile, desolventizing air temperature set is 250 DEG C, and desolventizing airshed 16L/min.Under positive ion and negative ion mode, capillary voltage is respectively+3kV and-3kV, and taper hole voltage is 0V.Taper hole pressure is 20psi(positive ion) and 40psi(negative ion).The mass charge ratio range of spectrum data collection is 50 ~ 1200 m/z, and the sweep frequency of collection is 0.25s.
3, XCMS collection of illustrative plates pre-service
UPLC-QTOF/MS serum positive ion ESI+ and negative ion ESI-detects the original Metabolic fingerprinting data of acquisition and is converted into Mzdata data file by the Masshunt software of Agilent company, then uses the XCMS software package of R language to carry outxCMScollection of illustrative plates pre-service, pre-service comprises retention time correction, peak identification, peak match, peak alignment, filter are made an uproar, Overlapped peak resolution, Threshold selection, standardization etc.xCMSpretreated correlation parameter is: peak half waist peak width is 10 (fwhm=10), and retention time window is set to 10 (bw=10), and other parameters are default value.xCMSobtain the two-dimensional matrix that can be used for statistical study after collection of illustrative plates pre-service, wherein every behavior sample (observation), be often classified as metabolin (variable), matrix intermediate value is corresponding metabolite concentration.And each metabolin peak use retention time (retention time, RT) and mass-to-charge ratio (mass-to-charge ratio,m/z) qualitative.Then this two-dimensional matrix uses R software packagecAMERAcarry out metabolin peak mark (comprising isotopic peak, adduct and fragmention).Carry out standardization to sample before statistical study, retention time range set to be analyzed is 0.5 ~ 10 min.Through the pre-service of XCMS collection of illustrative plates, in the data matrix that the UPLC-QTOF/MS spectrum of positive ion detecting pattern generates, comprise 522 metabolin peaks, anionic textiles pattern is 212 metabolin peaks.
4, LC-MS quality control of the experiment
When blood serum sample carries out metabolism group detection, arrange the order of 1 QC to insert in analyzing samples equably by every 10 analyzing samples in the QC sample of preparation, thus Real-Time Monitoring is from Sample pretreatment to the quality control situation pattern detection process.The original Metabolic fingerprinting of gained, after the pre-service of XCMS collection of illustrative plates, calculates the %RSD value (coefficient of variation) of each metabolin in QC sample, and with %RSD value for the longitudinal axis, and be that transverse axis is drawn (see figure 1) with retention time, the round dot in figure represents metabolin.Can the %RSD value of most metabolin as seen from the figure control below 30%, illustrate that Sample pretreatment is all right to the quality control in sample product testing process, the metabolism group data obtained be genuine and believable.
5, the metabolic profile preanalysis of Based PC A
The two-dimensional matrix data Random assignment obtained is become 2/3 as training sample training data(125 NEG:303 POS), other 1/3 as external testing sample test data(62NEG:151POS).Training sample is used and comes classification trend preliminary between observation group and outlier without supervision analytical approach and principal component analysis (PCA) (principal component analysis, PCA), see Fig. 2.In figure, the repeatability of QC sample can show that LC-MS quality control of the experiment is good.From figure, it can also be seen that between the examination positive (i.e. ill serum sample) and examination feminine gender (i.e. healthy serum sample), to there is certain classification trend, but still have partial intersection, need to adopt supervised learning method to realize further classification.
6, based on the metabolic profiling analysis of PLS-DA
For the deviation that gap in elimination group of trying one's best causes, obtain trend of comparatively significantly dividing into groups, the difference and the classification trend that have supervision analytical approach and partial least squares discriminant analysis (partial least squares-discriminant analysis, PLS-DA) to show the metabolic profile of the positive person under inspection of iodine staining and the negative person under inspection of iodine staining is used further for training sample.As shown in Figure 3, there is classification trend between metabolic patterns difference and obvious group, the R2X=0.231 of its modeling, R2Y=0.749, Q2cum=0.638 between the positive group of iodine staining and negative group.As can be seen from Figure 3, compared to the negative group of completely healthy iodine staining, be esophagitis, atypical hyperplasia or even more serious carcinoma in situ and the early stage cancer of the esophagus (the positive group of iodine staining) all occurred obvious metabolism 7,the PLS-DA model external certificate of cancer of the esophagus early screening
External testing sample two-dimensional matrix data (62NEG:151POS) is substituted in the PLS-DA model of above-mentioned foundation, obtain factor 1-3(and t [1], t [2], t [3] value of prediction), external testing sample is drawn in PLS shot chart, sees Fig. 5.And according to the feminine gender of the external testing sample shown in PLS-DA model and the positive, do ROC tracing analysis with actual classification label (examination is negative and positive), see Fig. 6.Wherein ROC area under curve AUC value is 0.99 (95%CI:0.978 ~ 1), and sensitivity is 95.4%, and specificity is 100%, and the rate of striving for of this model external testing sample predictions is 94.8%.This shows, the PLS-DA model prediction accuracy of the cancer of the esophagus early screening set up according to the inventive method is high, has good extrapolation effect.
The optimum prediction boundary value (cutoff) of the cancer of the esophagus early screening PLS-DA model of the inventive method is shown in Fig. 6 for 0.50(), this boundary value can be used in actual applications as the standard judging screening results, optimization and the balance of sensitivity and specificity can be reached, and other boundary values are not all optimum, sensitivity or specificity can be lost.
8, contrast experiment
In model construction research process, have selected the most significant metabolin of 10 species diversity according to the pretreated two-dimensional matrix of collection of illustrative plates, and with each difference metabolin for criterion, react cancer of the esophagus positive events by its content difference.Result shows, and the prediction level of each single metabolin is all not high, with the matching degree of actual conditions less than 89%, does not have extrapolation effect.
9, conclusion
As can be seen from above checking, only have the model built by the inventive method just can have good prediction effect.Gained PLS-DA model of the present invention is dangerous without over-fitting, and has classification trend between obvious group, can be good at healthy serum and suspected lesion serum to make a distinction by blood serum metabolic situation, sensitivity and specificity high, may be used for cancer of the esophagus early screening.
When adopting model of the present invention to carry out the application of cancer of the esophagus early screening, step is as follows:
(1) gather serum to be checked, pre-service is carried out, in order to sample detection to serum in step (1)-(4) in centrifugal rear employing above-mentioned 2.2;
(2) by pretreated serum sample to be checked according to above-mentioned 2.3 step carry out LC-MS detection, obtain original Metabolic fingerprinting;
(3) original Metabolic fingerprinting is carried out collection of illustrative plates pre-service according to the method for above-mentioned steps 3, obtain the metabolin information of this serum to be checked;
(4) this metabolin information is imported in PLS-DA model, calculate t [1], t [2], t [3] value, the prediction probability (be greater than 50% and be the positive) of cancer of the esophagus early screening risk can be obtained, thus judge cancer of the esophagus examination risk by body metabolism situation.In actual applications, if the metabolin information of serum sample to be checked is positioned at ill serum sample group region (the i.e. positive region of model, namely prediction probability is greater than 50%), then represent that this people has the ill danger of the cancer of the esophagus, indicative biopsy under needing to carry out further scope; If the metabolin information of serum sample to be checked is positioned at the healthy serum sample group region (i.e. negative areas, prediction probability is less than 50%) of model, then represents that this people does not have the ill danger of the cancer of the esophagus, do not need further to check.
In addition, in order to accelerate screening efficiency, the serum sample of many people can be gathered simultaneously, and be numbered, carry out LC-MS detection, collection of illustrative plates pre-service and data importing by disposable for multiple sample.
Non-limiting for the description to patent of the present invention above, based on other embodiments of patent thought of the present invention, all among scope.