Analysis And Study On Prognostic Factors Of Pediatric ALL Patients With MLL Gene Rearrangement

At present,the cure rate of acute lymphoblastic leukemia(ALL)in children is quite high,but some children are still refractory or easy to relapse.Pediatric ALL patient with MLL gene rearrangement has a poor prognosis.Studies have shown that some prognostic factors,such as,age,fusion partners,early therapeutic response did impact on OS of leukemia patients with MLL gene rearrangement.With the rapid development of Next Generation Sequencing Technology,RNA-seq technology is gradually applied to the genetic diagnosis of leukemia,and it is extremely important for accurate identification of poor prognostic genes.This study analyzed the prognostic factors of pediatric ALL patients with MLL gene rearrangement with clinical characteristics,early therapeutic response and differentially expressed genes,in order to provide a theoretical basis for further stratification and individualized treatment of MLL-rearranged leukemia.Part ⅠThe therapeutic efficacy of pediatric ALL patients with MLL gene rearrangement treated with CCLG-ALL2008 protocolObjective:To investigate the therapeutic efficacy and prognostic factors of pediatric ALL patients with MLL gene rearrangement treated with CCLG-ALL2008 protocol.Methods:During the period from 2008 to 2015,227 ALL cases were treated with CCLG-ALL2008 protocol for high-risk(HR)group,including 30 MLL+cases,24 MLL-BCR/ABL+cases,173 MLL-BCR/ABL-cases,197 MLL-cases.Overall-survival(OS)and relapse-free-survival(RFS)were calculated for positive(MLL+)and negative(MLL-)in HR groups.The clinical characteristics,response to treatment,and the survival were analyzed retrospectively,and the clinical outcome was compared between patients with and without MLL rearrangement.Survival was analyzed by long-term follow-up.Results:1.Compared with MLL-groups,the cases younger than 2 years groups with MLL+were much increased.Compared with MLL-BCR/ABL-and MLL-group,Non-M1(primitive+immature blasts<5%)of bone marrow evaluation on day 15,group with MLL+was much decreased.The number of cases with CR on Day 33 in MLL+ group was significantly higher than that in MLL-BCR/ABL+group.However,the number of cases with MRD ≥1×10-2 on Day 33 in BCR/ABL+group was significantly higher than that in MLL+group.Cases with MLL-BCR/ABL+group had a poor 10 years OS and RFS than MLL+group.2.Age did matter in MLL+group.Patient younger than 2 years old had a poor 10 years OS and RFS than those older than 2 years cases.Cases insensitive to prednisone had a poor 10 years OS and RFS than sensitive to prednisone.Cases NR group on D33 had a poor 10 years OS and RFS than CR group.Multivariate COX regression analysis found that age,MLL fusion partners,or prednisone response on day 8 has impacted on RFS.Age or prednisone response on day 8 has impacted on OS.Conclusion:Younger than 2 years of age,MLL/AF4,non-sensitivity to prednisone and D33NR might be the main factors of treatment effect of CCLG-ALL2008 protocol for HR group.Younger than 2 years of age,MLL/AF4,prednisone resistance and D33NR might be the poor prognostic parameters in predicting the outcome in childhood ALL with MLL gene rearrangement.Part ⅡPreliminary study on the relationship between differentially expressed genes and prognosis of pediatric ALL patients with MLL gene rearrangement by RNA-seqObjective:To identify MLL genes and the partners by RNA-seq technology,and to analyze the molecular markers associated with poor prognosis of pediatric ALL patients with MLL gene rearrangement by using the data of differentially expressed genes.Methods:1.From April 2014 to April 2018,15 cases of pediatric ALL patients with MLL gene rearrangement and 5 negative cases were enrolled in the study at the Children’s Hospital of Soochow University.RNA-seq was used to identify gene expression and identify partner genes.2.Differentially expressed genes(DEGs)were analyzed between different groups using Cuffdiff algorithm,and GO functional enrichment and KEGG pathway enrichment were analyzed between different groups.The prognostic genes were screened by analyzing the genes with the most significant differences in the top 20 ones which enriching to the meaningful GO functional enrichment and KEGG pathway enrichment.Results:1.The MLL gene and the partners were all identified in 15 cases by RNA-seq technology.2.There were significant differentially expressed genes between the MLL rearrangement positive group versus negative group,the relapsed group versus continuous remission group and the prednisone resistant group versus sensitive group.Build on the results of meaningful GO functional enrichment and the KEGG pathway enrichment,CCNA1、LAMP5、CXCL8、MAP7、IL1RL1、NTRK1、PROM1 were screened from the top 20 up-regulated DEGs of the MLL rearrangement positive group.In the relapsed group,IL1RL1、GATA2、IL5RA、CXCL8、CXCR2 and CXCR1 were selected as poor prognostic genes from the top 20 up-regulated DEGs which enriched into the meaningful GO function and KEGG pathways.Conclusion:1.Identification of fusion genes is very precise by RNA-seq technology which is expected to be a novel method for molecular biology diagnosis of leukemia.2.High expression of CXCL8,IL1RL1,CCNA1,LAMP5,PROM1,GATA2,CXCR2 and CXCR1 may be associated with poor prognosis of pediatric ALL patients with MLL gene rearrangement.