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Background and Purpose: Wilms' tumor is one of the most common malignant tumor in children, and children under 15 worldwide incidence of approximately 1/10, 000. The German physician Wilms 1899 first reported the disease, so most of the authors have called Wilms tumor. Wilms' tumor is the most common in children by renal blastema malignancies, the highest incidence in the age of 2. Cancer mortality in children has increased by 8% in the 1980s to 10.7%, the pediatric kidney neuroblastoma dead children has become one of the main reasons. Wilms tumor clinical pathologic staging and treatment choices have a direct relationship to the children prognosis, NWTS mentioned in its 2001 summary: (rely CT) imaging technology conditions still 7.0 % rate of misdiagnosis, Wilms tumor diameter less than 3cm. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry (Surface Enhanced Laser Desorption / Ionization time of flight Mass Spectrometry, SELDI-TOF-MS) is a new protein research technology developed in recent years, with fast, simple, high-pass volume and multiple parallel test advantages of mass spectrometry and bioinformatics methods appear to provide a specific marker for effective screening method. Ciphergen's SELDI-TOF-MS technology research and development is a new proteomics research methods, this technology through the application of a small amount of natural protein extract to quickly overcome the limitations of traditional analytical methods. Sample amount is small, easy to operate, high sensitivity, high-throughput, etc., have been successfully applied to ovarian cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer and other malignancies diagnosis and screening of tumor markers and other proteins genomics research. This group to apply the technology to get satisfactory results in liver cancer, thyroid cancer and renal parent cell tumor of preliminary studies. The application of SELDI-TOF-MS combined with bioinformatics and support vector machine (support vector machine, SVM) method, the screening of children with Wilms 'tumor-specific protein markers, establishment of Wilms' tumor clinical stage serum protein fingerprint model were compared with CT staging. Materials and methods: (1) serum samples from a total of 80 cases were from the First Affiliated Hospital of Zhengzhou University, Pediatric Surgery, of which 30 cases of Wilms tumor before surgery, 30 cases of other pediatric abdominal malignant solid tumors (16 cases of neuroblastoma, liver 4 cases of neuroblastoma, four cases of renal rhabdomyosarcoma, malignant teratoma four cases, pancreatic blastoma two cases), and normal control group of 20 patients were healthy children from physical examination. All malignant tumors were confirmed by immunohistochemistry and professor of pathology confirmed more than two. Wilms tumor before surgery, 21 males and 9 females, mean age 2.10 ± 0.10 years; pediatric abdominal malignant solid tumor were 18 males and 12 females, with an average age of 2.50 ± 0.16 years. Normal control group Wilms cell tumor group age-sex matched. All peripheral venous whole blood specimens during surgery before early morning fasting standing for 1h after 3000 rpm centrifugal 10min, 4 ℃ separated serum stored at -80 ° C low temperature refrigerator. CT projection method: Siemens SCT machine, scan range from the diaphragmatic 1cm above to kidney pole interval 8mm, thickness 8mm. Interested interval 5mm, thickness 5mm, scan time 2s. The contrast agent Omnipaque, the dose 1.5-2.0ml/kg. (2) samples of ice melt, 4 ℃ 10000 rpm centrifugal 2min; take a 96-well plate, put in the ice box, each hole plus U9 (9M Urea and 2% CHAPS, 1% DTT) 10 μl of serum 5μl, 4 ℃ chromatography cabinet oscillation at 600 rpm for 30 min; oscillation 15 min before the end to make the chip pretreatment, the chip into Bioprocessor in and write down the chip number; each well 200 μl of of plus NaAC (100 Mm, pH 4), chromatography cabinet 600 rpm, 5 min, repeat 1 times; U9 treatment after a 96-well plate put on ice to quickly join NaAC185μl chromatography cabinet shaking at 6000 rpm for 2 min; plus processing good sample 100μl to chip, chromatography cabinet 4 ℃ 600 rpm oscillation 1h, drying repeated 3 times; rapid drying of washed 2 times with HPLC water 200μl; repeated 2 times with 50% saturated SPA 1μl; on machine detection; the chip is placed in a the Ciphergen read spectrometer detection. (3) using a protein chip software 3.1 to do the correction, so that the strength and the molecular weight of the total ion homogenization. The Analyze the application ZUCI-Protein Chip Data System software package for the analysis. The raw data analysis using discrete wavelet (undecimated discrete wavelet transform) to remove noise, and subtract the baseline. Clustering analysis of the m / z difference of each sample is less than 0.3% to 10% as the minimum threshold, peak clustered into one class. Support Vector Machine (support vector machine, SVM) eigenvectors selection statistical filtering combined with model-dependent screening method discriminant model using leave-one-out cross validation as the evaluation model discriminant results. (4). Statistical analysis of all protein spectral raw data processing after the initial screening of all the mass-to-charge ratio peak Wilconxon rank sum test, to elect the smallest p-value 10 peak signal to noise and cluster analysis after filtering out further analysis. The effect of any combination of the 10 peaks (175 combinations) for input, support vector machine model using leave-one assessment model forecast, inspection standards set α = 0.01 1. The Wilms tumor I Wilms cell tumor, and Wilms cell tumor Ⅱ group Wilms tumor stage Ⅰ group and Wilms cell tumor Ⅱ period of mass spectrometry after preliminary filtering screening 483 M / Z peak, its relative strength to do the Wilconxon rank sum test analysis get 5 P values ??less than 0.01 M / Z peak from the significant differences in any combination of the protein peaks using SVM screened predictive value Youden (youden), the highest index of the combination model, screened M 3 / Z in 7965.4,5022.4 and 8469.6 markers, low expression in Wilms tumor II the stage Ⅰ high expression in Wilms' tumor. Potential markers as the union of two input values, with cross-examination of the leave-one-discriminant model on the test set, a sensitivity of 100% and a specificity of 100%. 2. I nephroblastoma Wilms tumor cell tumor, and Wilms tumor the III phase group Wilms cell tumor group Ⅰ and Wilms cell tumor stage Ⅲ 496 M / Z peak mass spectrometry after preliminary filtering screening, their relative intensity Wilconxon rank sum test analysis, P values ??less than 0.01 M / Z peak 4 from the significant differences in any combination of the protein peak, SVM screened predictive value (youden) Youden index the highest combination of model, filter out the M / Z 4 in 4263.1,4122.8,4330.7 and 4303.7 markers, low expression in Wilms tumor stage Ⅲ group, the stage Ⅰ high expression in Wilms' tumor. Potential markers as the union of two input values, using leave-one cross-examination, the discriminant model on the test set sensitivity of 100.00%, 100.00% specificity. 3. Wilms' tumor I renal neuroblastoma and Wilms cell tumor Ⅳ group Wilms cell tumor group Ⅰ and Wilms cell tumor Ⅳ of mass 565 M / Z peak after preliminary filter screening, their relative intensity Wilconxon rank sum test analysis, P values ??less than 0.01 M / Z peak 4 from the significant differences in any combination of the protein peak, SVM screened predictive value (youden) Youden index the highest combination of model, filter out the M / Z 2 in markers of 10836.6 and 8179.1, low expression in Wilms tumor IV of I high expression in Wilms tumor. The joint two potential markers as input values, with leave one out cross-validation, discriminant model on the test set sensitivity of 88.89%, and the specificity of 100.00%. 4. The Wilms tumor II Wilms tumor Wilms cell tumor stage Ⅲ group Wilms tumor group Ⅱ and renal neuroblastoma stage Ⅲ mass 490 M / Z peak after preliminary filter screening, their relative intensity Wilconxon rank sum test analysis, P values ??less than 0.01 M / Z peak 3 from the significant differences in any combination of the protein peak, SVM screened predictive value (youden) Youden index the highest combination of model, and filter out the M / Z two markers at 5019.2 and 4143.2, low expression in Wilms tumor stage Ⅲ group, high expression in Wilms tumor II. The joint two potential markers as input values, using leave-one cross-examination, the discriminant model on the test set sensitivity of 88.89%, and the specificity of 100.00%. 5. Wilms tumor II Wilms tumor Wilms cell tumor Ⅳ group Wilms tumor group Ⅱ and Wilms cell tumor Ⅳ of Mass Spectrometry 508 M / Z peak after preliminary filter screening, their relative intensity Wilconxon rank sum test analysis, P values ??less than 0.01 M / Z peak 20 from the significant differences in any combination of the protein peak, SVM screened predictive value (youden) Youden index the highest combination of model, filter out the M / Z markers located 7976.5 a low expression in Wilms tumor Ⅳ group, high expression in Wilms tumor II. Potential markers as the union of two input values, using leave-one cross-examination, the discriminant model on the test set sensitivity of 100.00%, 100.00% specificity. 6. Wilms cell tumor III Wilms tumor Wilms cell tumor Ⅳ group Wilms tumor group Ⅲ and Wilms cell tumor Ⅳ of the 504 M / Z peak mass spectrometry after preliminary filtering screening, their relative intensity Wilconxon rank sum test analysis, P values ??less than 0.01 M / Z peak 12 from the significant differences in any combination of the protein peak, SVM screened predictive value (youden) Youden index the highest combination of model, filter out the M / Z markers located 8194.4 a low expression in Wilms tumor Ⅳ group, high expression in Wilms tumor III. The joint two potential markers as input values, using leave-one cross-examination, the discriminant model on the test set sensitivity of 93.75%, and the specificity of 100.00%. 7. Ⅰ Ⅱ Wilms tumor Wilms cell tumor, and Wilms cell tumor III the phase group IV Wilms cell tumor Ⅰ Ⅱ phase group and Wilms cell tumor of III-IV of the mass spectrometer through a preliminary filter screening 519 M / Z peak the relative strength do Wilconxon rank sum test analysis P values ??less than 0.01 M / Z peak 3, from the significant differences in any combination of the protein peak, the highest the SVM predictive value of screening out Youden (youden), index of combination model screened 3 4153.9,3257.6 and 3290.7 M / Z is located markers in Wilms' tumor group Ⅲ Ⅳ period low expression and high expression in Wilms tumor Ⅰ Ⅱ period. Potential markers as the union of two input values, using leave-one cross-examination, the discriminant model on the test set sensitivity of 83.33%, a specificity of 93.75%. 8. The clinical staging control: Wilms tumor early diagnosis model of 2 m / z (6984.4,6455.5) analysis of serum markers the draw the Wilms tumor periods as follows: Ⅳ period relative to the low stage Ⅲ expression; Phase III for Phase Ⅱ low expression; Phase II for Phase I low expression; Phase Ⅰ low expression of normal children; latter relatively high expression; later clinical stage lower expression of the strength of the M / Z. 9. Blinded to verify the accuracy of the CT protein chip installments: protein chips installments as follows: Ⅰ 6 cases, 10 cases of stage Ⅱ, Ⅲ of 10 cases, Ⅳ stage 4 cases, 30 cases of Wilms' tumor periods with pathological installments consistent for compliance with both 100.00% control of CT staging with surgical stage: the period of surgery and pathology were confirmed by CT pathological stage Ⅲ patients included in Phase II, pathological stage Ⅳ 1 cases included in the Phase Ⅲ each period in line with the rate of 100.00%, 85.00%, 85.00%, 75.00%. Conclusion 1. m / z is located 5022.4 Da, 7965.4 Da, 8469.6 Da, 4303.7 Da, 4122.8 Da, 4330.7 Da, 4785.6 Da, 10836.6 Da, 8179.1 Da, 5019.2 Da, 4143.2 Da, 7976.5 Da, 8194.4 Da, 3290.7 Da, 4153.9 Da and 3257.6 Da protein composed SVM installments discriminant model accurate clinical staging of Wilms' tumor in children and the development of rational treatment programs are a good value. 2. Using SELDI-TOF-MS technology combined with SVM method to identify normal children and Wilms tumor I, II, Ⅲ, Ⅳ differential protein markers, they M / Z intensity is in turn reduce the periods in order of decreasing amplitude comparison significantly (P <0.05); normal children and Ⅰ high expression Ⅰ intensity lower than normal children; M / Z higher the intensity, staging the lower, the better the prognosis. 3. The blinded validation model: the protein microarrays staging than CT staging accuracy is consistent with the pathological findings, can make up for the shortcomings and deficiencies of CT staging on the biological characteristics of qualitative, build proteins specific marker surgery before more accurate the Wilms tumor clinical staging and qualitative, to make up for the lack of clinical laboratory examinations. 4. M / Z peak by mass spectrometry combined SVM derived, more precisely, from the thousands of peak filter out high specificity and sensitivity of the protein marker for the subsequent separation of the specificity of the Wilms' tumor protein and The purified provide a brand new idea and basis.
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