De Novo And Relapse Related Molecular Research Of Childhood B-lineage Acute Lymphoblastic Leukemia

Objective:Based on the next-generation sequencing(NGS)to capture and sequence the translocation of 2 chromosomes,then explore the correlationship between genomic fusion gene type and relapse in ETV6-RUNX1 positive childhood B-ALL.Investigate and verify the genetic variants which involved the “second hit” of childhood acute lymphoblastic leukemia,and initially clarify the pathogenic mechanism.Methods:In this study we established the NGS based ETV6-RUNX1 translocation capturing and sequencing method for 24 B-ALL children.Moreover,we compared patient-specific breakpoint and flanking sequence on paired diagnostic and relapsed samples.Through whole exome capture and next-generation sequencing,4 samples of a pair of ETV6-RUNX1 positive ALL monozygotic twins were tested,then GNAO1 was targeted as a focus to study,the function of mutated protein was predicted,the mutation site specific subclone and ETV6-RUNX1 fusion gene specific subclone evolutionary characteristics were analyzed.ETV6-overexpression lentivirus and ETV6-ShRNA were used to affect 293 T cell line,to show the influence on GNAO1 expression.Results:We captured patient-specific breakpoints in 24 cases of childhood ALL samples.Besides,we found the three-way translocation including ETV6-RUNX1 in 5 patients,and 4 of them relapsed,other 19 patients without three-way translocation were still in remission.Moreover,we also found that relapsed clone mainly derived from primary leukemia clone at diagnosis.We obtained the only mutation which was shared by the twins: GNAO1-R209 C.GNAO1-R209 C positive cells were subclone of ETV6-RUNX1 positive cells,and rapidly proliferated as the major clone when diagnosed in 2 months,and ETV6 inhibited the transcription of GNAO1.Conclusion:The three-way translocation may be an important risk factor of ETV6-RUNX1 positive leukemia relapse,moreover,next-generation sequencing could precisely obtain the genetic information of fusion genes,which could be benefitial to clinical examination.GNAO1 mutation was a “second hit” factor which involved in the occurance of ETV6-RUNX1 positive ALL.