Screening And Clinical Application Of Pediatric Neuroblastoma Serum Protein Markers

1 Background and objectiveNeuroblastoma(Neuroblastoma,NB)is the most common malignant solid tumors in children,8%-10% of all pediatric tumors,with early clinical symptoms of the disease is not obvious,the onset hidden,tumor growth is rapid,high malignant degree,early transfer characteristics,easily missed diagnosis and misdiagnosis and delayed treatment.At present,the early diagnosis of Neuroblastoma method is still with color doppler ultrasound,CT and MRI is priority to.The effect of early radical resection cooperate with postoperative radiation and chemotherapy is basic satisfactory.,but after entering the stage Ⅲ or IV,NB survival rate significantly decreased,and the recurrence rate is high.Resection degree of judgment and the tests of tumor recurrence are still lack of specificity.Based on the NB of the importance of early diagnosis and early treatment,urgent clinical needs a high sensitivity and accuracy of diagnosis and monitoring methods.Proteomics has been widely used in cancer research,especially tumor specific protein markers found ceaselessly,improving the level of early diagnosis and early treatment of malignancy greatly,and provide research direction for tumor therapy.This team has worked with the Chinese academy of sciences institute of biophysics and the tumor research institute of zhejiang university to use the relevant technical of proteomics to screen and identified the serum specific protein markers of renal tumor,breast cancer,gastric cancer and other malignant tumors successfully.The renal tumor specificity protein polypeptide has been synthesised successfully in vitro and the experiments in animals in vitro phase has gaind satisfactory curative effect.This topic is based on the early stage of the technical route,through to the normal group,the detection of serum tumor group,select specific protein markers,the different stages of children in detail group to verify again and statistical analysis,preliminary stage diagnosis model is established,combined with follow-up and surgical results,will filter out protein markers used in the prediction of tumor recurrence and the surgical removal of the degree of judgment.Finally through the quantitative analysis and compared with clinical common check methods,clear the sensitivity and specificity of clinical application.2 Materials and methods 2.1 Experimental material 2.1.1 Material of clinical casesThis topic serum samples study selected 365 cases were from the First Affiliated Hospital of Zhengzhou University in Henan Province,acquisition time in January 2008 to December 2013.Divided into: normal control group,tumor group(I stage,II phase,Ⅲ phase,IV stage),recurrence group,long-term survival group,palliative resection group,the radical resection group.The pathological diagnosis of children with Neuroblastoma experimental results are: Neuroblastoma(NB),all samples were confirmed by pathological biopsy or surgical resection made,were sent to our hospital pathology checks in accordance with uniform procedures,all the pathological diagnosis have been tested more than two hospital pathology experts.Blood sampling conditions: early morning 6:00,all children with Neuroblastoma that is both healthy children in the fasting state blood specimens from peripheral blood 5ml,at room temperature for 1 hour.After the centrifuge go to treatment,low temperature at 4 ℃ centrifuged for 20 minutes at ambient,speed: 3000 r / min,the centrifugal force: 3000 × g,the supernatant was extracted specimens to each tube 100 ul be dispensed,dispensing has been completed into;cold storage freezer,refrigerator temperature conditions:-80 ℃.In this study,subjects were obtained guardian consent,and approval by the ethics committee.2.1.2 The main instruments and reagentsUrea(Urea),DTT(DL-Dithiothreitol,1,4-dithio threitol),IAM(Iodoacetamide,iodoacetamide),CHAPS(3-cyclic amine 1-propanesulfonic acid),SPA(sinapic acid,erucic acid),TFA(trifluoro-acetic acid,trifluoroacetic acid),ultra-pure water(HPLC grade)and acetonitrile(acetonitrile,CAN)purchased from Sigma,Origin: USA;Spectra Multicolor Low Range Protein Ladder available from Thermo company,Origin: USA;Trypsin was purchased from Promega company,Origin: USA;Profiling Kit 100 MB-WCX available from Bruker Inc company,Origin: Germany.Protein Chip,WCX2 Protein Chip,PBS II + SELDI-TOF-MS and Bio-processor available from Ciphergen Biosystems company,Origin: USA;MALDI-TOF-MS available from Bruker company,Origin: Germany;High performance liquid chromatography,available from Shimadzu Corporation,Origin: Japan;2D-LC-LTQ-MS available from Thermo Electron Corporation,Origin: USA;ZUCIPDAS from Zhejiang University,Origin: China.2.2 Experimental methods 2.2.1 Construction pediatric Neuroblastoma serum protein markers for screening and clinical applicationsThe specimens have been thawed serum samples in an ice bath for 30 to 60 minutes,after complete thawing at 4 ℃ using a centrifuge and centrifuged for 5 minutes,and centrifuged at: 12000 × g,the supernatant was centrifuged specimens out of standby.5μL of serum samples,10μL MB-WCX Magnetic Beads,10μL MB-WCX Binding Buffer are then added to an Eppendorf tube,thoroughly shaken,implantable beads separator,after joining MB-WCX Wash Buffer 100μL,MB-WCX Elution Buffer 5μL added Eppendorf tubes containing beads,repeated pipetting,MB-WCX Stabilization Bu ffer 5μL added to the test tube and pipette again and set aside.Chip pretreatment.Adding diluent U9(1% DTT,9mol/L urea,2% CHAPS),mixed and shaken,weak cation exchange was added chip holes,96 samples by weak cation exchange WCX2 Protein Chip disposable chip to be detected,WCX2 Protein chip inserted Bio-processor work holder,carefully recording the coordinates of the situation of the chip.Import SELDI-TOF-MS instruments to detect differences of serum protein screening.Organize and Export SELDI-TOF-MS assay data,invalid data is removed,the molecular weight and the relative expression of the standard should be the same,complete extraction of each sample protein molecular weight and the relative expression of accurate data.SELDI-TOF-MS data extracted through ZUCIPDAS technical analysis(Zhejiang University),and strictly exclude interference processing technology,selected m/z difference is less than 0.3% of the data,and classified as a class.Wilcoxon rank sum test for screening for further processing and analysis of data screening and treatment of different groups of data,extraction of m/z identical,m / z peak corresponding to different data for differences in proteins by SVM screened to give combined model,youden highest index.Children draw Neuroblastoma specific serum protein markers,statistical analysis of serum samples of different stages of protein markers,draw the corresponding expression intensity data.The recurrence group,the long-term survival group,different serum samples extent of surgical resection group of children in the differentially expressed protein markers for statistical analysis,the expression of different groups of children with protein markers.2.2.2 Purification and identification of children with Neuroblastoma serum protein markersApplication of HPLC separation and purification of the target protein in a sample,separation and purification of the object based on SELDI-TOF-MS screening results,select from serum samples before a higher target protein expression of tumor surgery group.Ice water bath thawed serum samples taken from-80 ℃ refrigerator,drawn 100μl serum samples.600μl ACN added ultrapure water and 300μl 100μl serum samples,pipetting,centrifugation,SPD SpeedVac vacuum centrifuge enrichment cooling system and the supernatant was lyophilized,HPLC purified rinsed C18 column above serum sample,EP tube collection corresponding to the peak in the HPLC chart proteins using MALDI-TOF-MS protein sample mass to charge ratio detection,and to find the mass to charge ratio sample 5920 Da similar protein is located(approximately 0.03% of the errors exist).Enzymatic target protein.Series 2D-LC-LTQ-MS system,spray system and sample after elution column for peptide mass to charge ratio spectrum detection to give the target protein.SEQUEST application program data retrieval target protein,Bioworks database search,retrieve and peptides and amino acids may match resulting protein.2.2.3 Validation of the pediatric Neuroblastoma serum protein markers and contrast sensitivity and specificityELISA methods of target protein(Class APO C-Ⅲ)were quantitatively analyzed.Lyophilized standards with Class APO C-Ⅲ,increasing concentrations of each 2-fold.Pipette calibration,take the 100μl sample of each concentration,the tip vertical drop added 96,ahead of anti-Class APO C-Ⅲ antibody pre-coated,and set three wells.Each group were 10 serum samples taken 100μl of each concentration of the sample,the tip vertical drop added 96,closing the entire plate with Closure plate mold,thermostat control in strict accordance with the instructions incubation,microtiter plates.After each hole to absorb the liquid,anti-Class APO C-Ⅲ antibody labeled with biotin,take 100μl to the wells,microtiter plates were incubated again;plate with tap water and detergent filled,glass,electrophoresis tank and related accessories to be cleaned,in pat dry with absorbent paper hole liquid,the use of a hood,the natural state dry.ABC working solution was added to each well after the plate fully dry,microtiter plates are incubated;ultra-pure water again washer,dry natural state,90μl TMB color reagent was added to each well microtiter plate after incubation each added 100μl TMB Stop solution.Adjustment microplate reader to 450 nm,in each well and the absorbance read color-terminated within 15 min,the absorbance value of the standard curve plotted standard,and then calculate the concentration of the sample.ELISA method of target protein(Class APO C-Ⅲ)and the results of quantitative analysis commonly used in clinical diagnostic methods were compared to pathologic findings as the gold standard of judgment to validate the sensitivity and specificity of the diagnostic model Class APO C-Ⅲ.3 Result 3.1 Screening children Neuroblastoma serum protein markersStandardized and analyzed for tumor and normal group mass spectral data,information extraction peptide peak data peak data and the breakdown of proteins protein expression.Wilcoxon rank sum test for protein peak data processing(P<0.01),through data analysis and statistics to give their protein expression of m/z its peak;data and comparison of the two t-test(α=0.01),for the above two sets of data statistical analysis: protein expression Neuroblastoma serum peak has six,serum protein peaks Neuroblastoma have a low expression by SVM screened to give a combination of models,youden highest index,results: m/z 5920 Da marker protein.The marker in the normal group with low expression,the expression intensity 132.87 ± 93.41;highly expressed in the tumor group,the expression intensity of 760.42 ± 414.25.Two sets of data difference was statistically significant,P<0.01.3.2 Pediatric Neuroblastoma serum protein markers M/Z 5920 Da to determine tu-mor stageApplication of the above screening technique to express the intensity of NB different stages m/z 5920 Da of comparative analysis,the results suggest: NB-I of the group,NB-II of the group,NB-Ⅲ of the group and the NB-IV of group m/z 5920 Da expression of the protein were 422.58 ± 120.81,663.37 ± 219.40,930.64 ± 278.69,1229.59 ± 338.85.While the normal group,NB-I of the group,NB-II of the group,NB-Ⅲ of the group and the NB-IV of the group were analyzed statistically,the difference between any two sets of data were statistically significant,P<0.01.3.3 Pediatric Neuroblastoma serum protein markers M/Z 5920 Da predict tumor recurrence and judgmentOf m/z 5920 Da protein markers in front of the normal group,Preoperative group,relapse group,long-term survival group differences were analyzed,the expression of strength as follows: normal group was 178.98 ± 129.82,preoperative group was 1325.61 ± 471.70,recurrence group was 1325.61 ± 471.70,long-term survival group was 225.16 ± 165.08.m/z 5920 Da protein marker in relapse group comparison with Preoperative group,the difference was not statistically significant(P>0.05),comparing with the normal group,the difference was statistically significant(P<0.01);m/z 5920 Da protein markers in long-term survival group comparison with the normal group,the difference was not statistically significant(P>0.05),comparing with the preoperative group difference was statistically significant(P<0.01).3.4 Pediatric Neuroblastoma serum protein markers M/Z 5920 Da to deter-mine the degree of invasive surgeryDifferentially m/z 5920 Da protein markers in various invasive surgery degree groups were compared,the expression of strength as follows: normal group was 178.98 ± 129.82,preoperative group was 1125.00 ± 577.57,palliative resection group was 1054.02 ± 417.01,radical resection group was 625.44 ± 338.57;m/z 5920 Da protein marker in palliative resection group comparison with preoperative group,the difference was not statistically significant(P>0.05),comparing with the normal group,the difference was statistically significant(P<0.01);m/z 5920 Da marker protein expression in comparison with the normal group and radical resection group,the difference was not statistically significant(P>0.05),comparing with the preoperative group difference was statistically significant(P<0.01).3.5 Purification and identification of pediatric Neuroblastoma serum markersApplication of HPLC separation and purification of the target protein in a sample,EP tube collection on the HPLC chart corresponding to the peak of the proteins to make MALDI-TOF-MS detection,identify mass to charge ratio 5920 Da protein or peptide samples.Digested mass to charge ratio of proteins or peptides 5920 Da,into 2D-LC-LTQ-MS detection system,access PMFs,further testing analysis to obtain enzyme protein fragment amino acid sequence,import SEQUEST,application Bioworks database searching and matching obtain the complete amino acid sequence of FIG.Mass to charge ratio of 5920 Da protein or peptide having the sequence: KDALSSVQESQVAQQARGWVTDGFSSLKDYWSTVKDKFSEFWDL DPEVRPAS,this peptide is APO C-Ⅲ(Apolipoprotein C-Ⅲ).3.6 Verify children Neuroblastoma serum protein markersThe stage assay by ELISA class APO C-Ⅲ content pediatric Neuroblastoma different stages,through statistics and data analysis,the results suggest that with the rising stage,class APO C-Ⅲ content increased in turn,normal group,I stage,II phase,Ⅲ phase,IV phase of APO C-Ⅲ expression levels were 545.55 ± 176.09 pg/mL,873.55 ± 212.59 pg/mL,1691.64 ± 837.04 pg/mL,3155.64 ± 495.86 pg/mL,4633.91 ± 504.01 pg/mL,pairwise differences were statistically significant(P<0.01).The present phase of the experiment by ELISA for the normal group,the preoperative group,relapse group and long-term survival group,the quantitative analysis of class APO C-Ⅲ,expression levels were 545.55 ± 176.09 pg/mL,3037.33 ± 1448.09 pg/mL,4428.09 ± 1255.57 pg/mL,545.91 ± 289.26 pg/mL,recurrent group and the normal group difference statistically significant(P<0.01),the preoperative group and long-term survival group difference was statistically significant(P<0.01).The present phase of the experiment by ELISA for the normal group,the preoperative group,palliative resection group,the expression of radical resection group class APO C-Ⅲ quantitative analysis,expression levels were 545.55 ± 176.09 pg/mL,3037.33 ± 1448.09 pg/mL,3166.18 ± 958.38 pg/mL,1814.64 ± 571.74 pg/mL,palliative resection group Compared with normal group difference was statistically significant(P<0.01),radical surgery group Compared with preoperative group difference was statistically significant(P<0.01).3.7 Pediatric Neuroblastoma serum protein markers of sensitivity,specificity verificationThis phase of the experiment with the ELISA method for quantitative analysis of samples class APO C-Ⅲ to the result as a clinical diagnostic model,commonly used in clinical and laboratory examinations means of comparison,such as: 64 scan CT 、3.0MRI,to pathology as a result of Analyzing the gold standard to validate the sensitivity and specificity class APO C-Ⅲ diagnostic model,which is part of the experiment subjects were selected from the blind children,compared with healthy children,sensitivity class APO C-Ⅲ diagnostic model was 96.0%,specificity of 100%,and relatively common pediatric abdominal solid tumor sensitivity class APO C-Ⅲ diagnostic model was 96.0%,specificity of 88.0%.4 Conclusions4.1.The target protein m/z 5920 Da as pediatric Neuroblastoma specific serum biomarkers for clinical suspicion Neuroblastoma,can detect the protein markers.4.2.The target protein m/z 5920 Da as serum biomarkers in children with Neuroblastoma staging of sarcoma,recurrent tumors early warning and determine the extent of surgical resection of important guiding significance.4.3.By the target protein or peptide statistical analysis and identification proteins or peptides m/z 5920 Da initially identified as class APO C-Ⅲ.4.4.Class APO C-Ⅲ than other commonly used diagnostic imaging method has obvious advantages in the early diagnosis of children with Neuroblastoma sensitivity.4.5.Class APO C-Ⅲ Neuroblastoma in children with common pediatric abdominal neoplasms entities better specificity,suggesting that class APO C-Ⅲ may be present in a variety of solid tumors in children.The latter part of the study shall further expand the sample size,compared with other tumors,in order to clarify its specificity.Meanwhile,to further explore the relationship between class APO C-Ⅲ and the development of NB,which clearly class APO C-Ⅲ key role in the pathogenesis of NB.