Aberrant CpG Island Methylation in AML Is Associated with Disease Progression and Relapse.

Abstract

The role of DNA methylation in relapse and progression of acute myelogenous leukemia (AML) is incompletely understood. We studied DNA methylation of 9 promoter-associated CpG islands of genes frequently hypermethylated in leukemic cell lines. These were NOR1, NPM2, HIN1, SLC26A4, CDH13, PGRA, PGRB, OLIG2 and the tumor suppressor gene p15INK4b. We examined bone marrow and/or peripheral blood cells collected at the time of diagnosis and at the first relapse from 32 patients (13 females, 19 males) with AML. The median age was 58 years (20–68), the median survival was 18 months (8–80), the median blast count was 64% (20–98), and 10 patients had additional solid tumors and/or lymphatic/hematologic malignancies. Bisulfite treatment of DNA, followed by PCR and pyrosequencing were used to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands. We analyzed methylation data for individual genes and for a methylation index derived after Z-score (z = [value –mean]/standard deviation) transformation to equalize absolute differences between individual genes and we used paired t-tests for statistical analysis. Abnormal hypermethylation (≥10%) in bone marrow and peripheral blood cells at diagnosis was detected in all 9 investigated genes, with a range of 7/29 (24%) for HIN1 to 24/32 (75%) for CDH13. On average, an increase in methylation between diagnosis and relapse was detected in all genes, and was significant for CDH13 (mean 10%, p=0.0006), SLC26A4 (mean 7%, p=0.0012), HIN1 (mean 8%, p=0.0037), NPM2 (mean 7%, p=0.0073), p15INK4b (mean 13%, p=0.0081), NOR1 (mean 4%, p=0.0124), PGRB (mean 6%, p=0.0144), PGRA (mean 9%, p=0.0275), and OLIG2 (mean 3%, p=0.0732). When analyzed by change in methylation status: negative (methylation below 10%) turning positive (methylation ≥ 10%) and vice versa, of 238 analyses, 39 (16%) showed a negative to positive switch, 15 (6%) showed a positive to negative switch, and the remaining 184 (77%) were either positive or negative unchanged. Finally, when analyzed in individual patients, an increase in methylation was noted in 29 of 32 patients (91%). The median increase in methylation index between diagnosis and relapse calculated as a delta-Z-score was 30% (range from −10% to 147%), and was highly significant (p<0.0001). In summary, abnormal hypermethylation in bone marrow and/or peripheral blood cells from AML patients was detected in all investigated genes at diagnosis. Methylation levels further increased at relapse of the disease in 29 of 32 patients in 1 to 8 of 9 investigated genes. Based on quantitative analyses, we propose that methylation of CDH13, PGRB, PGRA and OLIG2 CpG islands are early markers for AML, while hypermethylation of HIN1, NPM2 and p15INK4b CpG islands is associated with disease progression and predominantly appears at relapse. Thus, aberrant hypermethylation is clearly associated with disease progression and relapse in AML, and likely mediates drug resistance in this setting. Increase of Methylation Index in Relapse Increase of Methylation Index in Relapse