The Study About The Application Of Circulating Tumor Cells And UBE2C In Neuroblastoma

OBJECTIVE:The aim of this study is to establish and optimize a sensitive and specific method of isolating and identifying for circulating tumor cells?CTCs?in peripheral blood of children with neuroblastoma.Test the sensitivity and specificity of this method and evaluate the application value of this method on assessing the efficacy of treatment for neuroblastoma?NB?.Then perform bioinformatic analysis of neuroblastoma?NB?by Chip-analysis and Data-mining,screen the specific genes of different stages and construct the gene network.In order to further clarify the mechanism of stage?NB and provide theoretical foundation for the diagnosis and treatment of NB,the genes related to this stage was enriched and their function was analyzed.Select the gene UBE2C which is the key gene in the network as the research subject and detect the expression of UBE2C in neuroblastoma?NB?tissue and analyze the association with the clinical features of NB patients.Further verify whether it can serve as the tumor biomarker to predict the prognosis of neuroblastoma.?METHODS:1.Laboratory studies:1)Explore the expression of CD56,GD2 and CD45 proteins in neuroblastoma cells SH-SY5Y and IMR32 by immunofluorescence assay.2)Blood samples spiked with neuroblastoma IMR32 cells?range from 1-10 and 10-100?were enriched with CD56 magnetic beads positively and the CD56⁺cells were stained with antibodies labeled with fluorescence.Count the number of GD2⁺/DAPI⁺/CD45^-cells using fluorescent microscope and calculate the sensitivity of this method.3)Another 20 blood samples from non-tumor patients were sorted with CD56 magnetic beads positively and stained with antibodies labeled with fluorescence.Count the number of GD2⁺/CD45^-/DAPI⁺cells to test the specificity of this method.2.Clinical research:Children with stage?and stage?neuroblastoma in Department of Hematology and Oncology,Xinhua Hospital were recruited into our research group.Before chemotherapy,the detection of Circulating Tumor Cells,Minimal Residual Disease and the radiographic assessment were performed.The levels of neuron-specific enolase?NSE?in serum and vanilmandelic acid?VMA?in 24 hour urine were tested at the same time.These operations were performed again after 2 cycles chemotherapy.3.Data Analysis:The dataset of GSE16476,which included 88 cases of stage?to stage?s NB samples,was downloaded from the GEO database for bioinformatic analysis.After re-commenting by the R software Affy package,the limma package was used to screen the specific genes of different stages and the log₂?fold change??1 and FDR q-value<0.01 were set as the significant threshold.Weighted Correlation Network Analysis?WGCNA?was performed to construct the network of genes related to specific stages and the function of the network was analyzed by GO annotation and enrichment.4.Immunohistochemistry:Paraffin-embedded surgical tissue specimens from 60 NB patients kept by the Department of Pathology,Xinhua Hospital from January 2012 to June2015 were collected.The expression of UBE2C protein was detected by immunohistochemistry and the clinicopathological characteristics of these patients were analyzed retrospectively.The survival curve was established with Kaplan-Merier analysis,log-rank test,Chi-square test and Cox regression analysis were used to explore the correlation of UBE2C protein expression with clinicopathological characteristics and prognosis of NB patients.RESULTS:1)In neuroblastoma SH-SY5Y and IMR32 cells,there existed the expression of CD56 and GD2 proteins,while no CD45 protein was detected.In 1-10?10¹?IMR32 cells group,the average sensitivity of this method was79.92±6.73%;in 10-100?10²?IMR32 cells group,the average sensitivity of this method was 79.14±3.97%.No DAPI⁺/GD2⁺/CD45^-cells were detected in the blood samples of 20 non-tumor patients,so the specificity of this method was 100%.2)Now we have separate CTCs successfully from the peripheral blood of 9 patients,so the positive rate of CTCs was 75%.After 2 cycles chemotherapy,we found that the numbers of CTCs decreased to different extent and the change of CTCs was in accordance with the change of levels of serum NSE,24h urine VMA and Minimal Residual Disease.The results of radiographic assessment showed that 3 patients had partial response,6 patients had mixed response and 3 patients had no response.The change of CTCs can reflect treatment effect more sensitively than radiographic assessment.3)The gene expression spectrum of stageI-II and stageII-III was relatively stable.However,there existed substantial differentially expressed genes in stage-IV.By weighted correlation network analysis,we found a gene module?network?which was highly related to stage IV NB.Furthermore,we found this module was involving in cell cycling regulation after GO?Gene Ontology?enrichment analysis.4)Of 60 cases of NB,the male-to-female ratio was 1.73?1.The median age at diagnosis was 36months?range 1-156 months?and median duration of follow-up was 30.7 months?range 3.87-47.9 months?.The results of immunohistochemistry showed that the UBE2C protein positive expression rate in stage III and IV group?89.7%?was significantly higher than that of the stage I,II and IVs group?51.6%??P=0.001?;the UBE2C protein positive expression rate in poor differentiation group?82.9%?was also higher than that of the good differentiation group?52.0%???P=0.021?.The survival curve based on Kaplan-Merier analysis revealed that patients with UBE2C positive expression had poor prognosis?P=0.006?.The two-year overall survival?OS?of patients with UBE2C expression was 64.3% and that of patients without UBE2C expression was 100%.Both univariate and multivariate Cox regression analysis revealed that expression of UBE2C protein was independent prognosis factor for NB?P<0.001?.CONCLUSIONS:1?Immunomagnetic beads positive sorting combining with immunofluorescence staining techniques has relatively high capture sensitivity and specificity for neuroblastoma cell line IMR32 cells in the 3ml with?10² cells in the peripheral blood of non-tumor donors.2)The changes of the number of circulating tumor cells can reflect the treatment efficacy efficiently and show predictive value in advance toradiographic assessment.3)A large number of studies have certificated that disorders of cell cycle will lead to proliferation and metastasis of tumor,so the gene module for stage-IV NB found by our study will play an important role in exploring the mechanism of metastasis and progression of stage-IV NB.4)The expression of UBE2C protein was closely related with the stage and the differentiation of NB patients.Positive expression of UBE2C protein implied the poor prognosis for NB.UBE2C may play an important role in the invasion,metastasis and relapse of NB.It might become a new biomarker and potential drug target to NB.