Application Of Integrated Panel Testing Based On Next Generation Sequencing In Neuroblastoma

Objective:Neuroblastoma has a high degree of heterogeneity,from the self-regress to the rapid progress.Especially for high-risk group of neuroblastoma patients,even through multi-mode high-intensity treatment,the overall survival rate is still less than40%.It must be closely related to the complexity of its unique molecular biology,so the prognostic stratification and targeted therapy of neuroblastoma patients,based on variations of the genes and molecules,may be the best choice to solve these problems.The MYCN gene amplification is the best prognostic factor in neuroblastoma patients.However,more than 60% of children with neuroblastoma lack this change.Most patients are still lack of the evidences of based genetic changes to guide prognostic judgment and targeted therapy.It can be seen that the exploration of the genetic etiology of neuroblastoma is urgent.Methods:In this study,we used target-capture next-generation sequencing technology to capture the regions closely related to the occurrence and prognosis of neuroblastoma.Finally,a total exon regions of 53 genes and 3 large regions were designed,a total of212948 bp size.Then we applied this panel to test DNA from the fresh tumor tissue and matched peripheral white blood cells of 33 neuroblastoma patients without MYCN amplification confirmed by FISH in depth sequencing.SPSS19.0 software was used for statistical analysis through associating the sequencing results with clinical data.P values less than 5% were considered statistically significant.Results:The results showed that the mean sequencing depths of 887× across all target regions on all samples,and the maximum and minimum sequencing depths were1376 × and 438 ×,respectively.In addition,the unique depth was 474 ×.And the capture rate of most samples was very close to 70%,which indicated that the capture efficiency of the panel was very good.Overall,we identified 17 unique somatic aberrations in 11 patients,spanning 14 genes.The main changes were copy number variations,including 13 copy numbervariants(occurring in 8 patients): 2 amplifications in CDK4 and CCND1,1amplification in OS9,MYCN and DDX1,1 deletion in 11 q,CDKN1C,CDKN2 A,H19,RBMS3 and TIAM1.We also found 4 point mutations: 2 ALK(p.F1174L),1 BRCA2(p.K2392N),and 1 PHOX2B(p.F86fs).Among them,TIAM1 gene with its deletion was found for the first time in neuroblastoma.According to the results of copy number variations,33 patients with neuroblastoma were divided into CNV(+)and CNV(-)groups.There were significant differences between the two groups in INPC,INSS and distant metastasis(P = 0.002,P = 0.024 and P=0.038).The patients of CNV(-)group had a three-year EFS of 60.0% and CNV(+)was 16.7%(P = 0.015,HR = 0.1344,95%,CI =0.027~0.678).In addition,patients with CNV had poor response to conventional chemotherapy(P = 0.059).Conclusion:This study developed a panel related to neuroblastoma,and through a comprehensive quality control analysis,the result showed that the capture efficiency was high,and sequencing data had a high reliability.Based on our sequencing results of 33 patients by our panel,we found that CNVs were more common to be detected,which were positively correlated with UH,INSS 4 and distant metastasis,and 3-year EFS was significantly reduced in patients with CNV(+).In addition,patients with CNV had poor response to conventional chemotherapy.Therefore,CNV could be a predictor of poor prognosis in neuroblastoma.The deletion of TIAM1 gene,the new mutation we discovered,is likely involved in the differentiation process of neuroblastoma.Most CNVs are mainly involved in cell cycle,suggesting CDK4 / CDK6 inhibitors ccould be used for targeted therapy of neuroblastoma.The comprehensive genetic/genomic profile,by the method of deeply next generation sequencing,could compensate prognostic information and improve targeted therapy,and lay the foundation for the study of the pathogenesis of neuroblastoma.