DNA Methylation And Clinical Study Of Acute Myeloid Leukemia Based On Next Generation Sequencing

Objective:Acute myeloid leukemia(AML)is a clonal disorder of myeloid hematopoiesis and a predominantly fatal hematopoietic malignancy with high heterogeneity.Clinical and genetic features incompletely predict outcome in acute myeloid leukemia(AML).Aberrant DNA methylation is a common theme and a hallmark of AML.Recent studies on genome-wide DNA methylation have showed that aberrant DNA methylation was suitable for AML as a prognostic biomarker.However,the value of clinical methylation assays for prognostic markers has not been extensively explored due to the limitation of technology.Genome-CpGs-scale methylC-capture sequencing(MCC-Seq)is a new next generation sequencing(NGS)capture approach that interrogates functional methylomes based on the NimbleGen SeqCap Epi CpGiant system with a unique design and long probes.This study was conducted to detect the DNA methylation level of AML patients,to assess the prognostic implications of MCC-Seq in patients with de novo AML by integrating DNA methylation and genetic risk stratification,and to find genes regulated by DNA methylation associated with poor prognosis,thus to improve the prognosis stratification system for the precise diagnosis and treatment of AML,so as to improve the curative effect and the prognosis of these patients.Methods:Next-generation capture sequencing,MCC-Seq,assessed the DNA methylation level in 44 samples from 21 cases of de novo AML,2 cases of relapse AML,and 5 cases of healthy donor,who visited our hematology department between August 2014 and June 2016 were enrolled in the study.In MCC-Seq,a whole-genome methylation sequencing library is prepared with the qualified DNA samples,bisulfate-converted,and amplified,followed by a capture enriched for targeted bisulfite-converted DNA fragments.Raw sequence reads were processed to a series of bioinformatics and statistics analyses.The performance of MCC-Seq was evaluated by quality control and replicable experiments.The DNA methylation signature of different genomic features were evaluated by DNA methylation level index(DMI),and differentially methylated regions(DMRs)associated with prognostic genetic information were identified by comparisons of different cytogentic and molecular risk groups.Genes with promoter hyper-DMRs associated with worse genetic risk status were used to calculate a prognostic gene methylation summary value(M-value).Potential factors associated with the M-value were analyzed both in the study set and TCGA set.The association of M-value with clinical outcomes(i.e.,complete remission,overall survival,and disease-free survival)were independently validated in our study set(n = 21)and The Cancer Genome Atlas(TCGA)set(n = 169).Results:MCC-Seq exhibited good performance in AML patients with a high Q30 value and a stable repeatability.DNA methylation level was independent of patient characteristics.Promoters have major different functional DNA methylation signatures,which were associated with established genetic prognostic stratifications.A panel of 12 differentially methylated genes(DMGs)(i.e.BARD1,BCL9L,CLEC11A,DEFB1,FOXD2,IGF1,IL18,ITIH1,LSP1,P2RX6,RNASE3,and TUBGCP2)was identified with promoter hyper-DMRs associated with the outcome.A high M-value,compared with a low M-value,was associated with failure to achieve complete remission(40.0%vs.90.9%,p = 0.024),increased hazard for disease-free survival in the study set(HR:6.83,95%CI:1.07-40.28,p = 0.039)and poor overall survival in TCGA set(HR:1.491,95%CI:1.043-2.151,p = 0.038),For intermediate-risk AML(IR-AML),patients with a low M-value were more likely to achieve CR.Besides,hematopoietic stem cell transplantation(HSCT)and survival outcomes were not adversely affected by a high M-value(p = 0.271).Conclusions:MCC-Seq is a stable,reproducible,and cost-effective methylation assay in AML.A 12-gene M-value encompassing epigenetic and genetic prognostic information represented a valid prognostic marker for patients with de novo AML.