Aberrant Methylation Of DNA Promoter In Acute Leukemia And Its Clinical Significance In Detection Of Minimal Residual Disease

With the development of the study of Leukemia pathogenesis and treatment strategy, the complete remission rate of leukemia has been improved remarkably. Because of the minimal residual malignant cells, leukemia relapse is the most formidable point of treatment. Genetic and epigenetic changes are the important alterations found in acute leukemia. The unification of these two mechanisms applies for inhibiting tumor suppressor genes. As the most frequent alterations in AL, aberrant DNA Promoter methylation is the dominant mechanism for AL development. Aberrant methylation of a specific gene or a panel of genes may be the new molecular marker for AL prognosis and the target for AL treatment.In this study, we use bisulfate sequencing PCR (BSP) technologies to assess the DNA methylation status in bone marrow samples from different stages (diagnosis, CR and relapse) of AL and normal bone marrow. Aberrant methylation of ID4 and ZO-1 gene is a keeply phenomenon in these stages of AL. On this basis, quantity methylation level of ID4 and ZO-1 gene of patients with AL detected by methylight technologies. The sensitivity of this methods is 10-5.In addition to identification of the possibility of monitoring relapse and MRD by detecting methylation level of ID4 and ZO-1 gene, we study the correlation of serial AL patients' bone marrow with clinical outcomes, such as treatment effects and prognosis.The results indicated that:1, the expression of ID4 was detectable in all the 9 cell lines drived from leukemia by Real-time MSP, while onle 7 cell lines were ZO-1-positive by Real-time MSP. And what's more, the methylation level of cell lines drived from lymphocyte was highter than that from myeloid.2, the expression of ID4 and ZO-1 were negative in healthy donor bone marrow, however, the methylation rate of ID4 and ZO-1 were 72% and 29% in acute leukemia bone marrows, respectively. The methylation rates and expression levels of both ID4 and ZO-1 were highter in ALL than those in AML. However, ID4 was no specificity among the AML subtypes and consistant with genetic index, so it can be work as a pan-leukemia marker.3, the methylation level was close related with clinical oberservation under different patient status. The serial cases results showed that methylation level has a positive relation with leukemia load.4, the methylation level was consistant with known genetic index in different stages in the same patient, which confirm the feasibility of methylation level in leukemia detection.5, the minimal residual leukemia can be detected ahead 1-3 months in CR patients shown that it has the potential to be applyed in detection of minimal residual disease.In conclusion, this study successfully established method of qMSP with high specificity and sensitivity for ID4 gene and ZO-1, and first provides evidence to show that ID4,ZO-1 were an epigenetic tumor marker of AL. Demonstrated wide existence of ID4 gene and ZO-1 promotor methylation among various acute leukemia subtypes. The quantitative methylation level, might reflect the change of tumor load in acute leukemia patients. Different clones of leukemia cells had different methylation levels.This report Although it requires to be further authenticated in a large-scale and prospective study, the aberrant methylation of ZO-1 and ID4 could be potentially used as a biomarker for prognostic evaluation of AL patients.