Lack of Increased Gene Promoter Methylation at Relapse in Acute Promyelocytic Leukemia (APL).

Increasing methylation of gene-regulatory DNA sequences has been associated with the progression of malignancies, and, in APL, the pathogenetic PML-RARα fusion gene has been associated with DNA methyltransferase activity, which might contribute to this process. We, therefore, hypothesized that promoter methylation levels would be increased in APL samples at disease relapse. Preliminary studies of a few CpG-enriched gene promoters, using highly quantitative pyrosequencing technology, demonstrated considerable individual-specific variation in DNA methylation. Hence, we used matched pretreatment and relapse bone marrow and/or peripheral blood samples with >85% promyeloblasts after low-density gradient enrichment from 4 APL patients. High quality DNA was prepared from fresh samples by a guanidinium-cesium chloride gradient procedure. Global analysis of CpG-enriched DNA was performed by the HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay (Khulan, et al, Genome Res. 16, 1046Khulan, et al, Genome Res. 16, 2006), using a microarray representing the promoters of 24,000 human genes. The HELP assay relies upon a comparative genomic hybridization of HpaII (methylation-sensitive) versus MspI (methylation-insensitive) digested genomic DNA and is able to distinguish populations of methylated loci from hypomethylated (HO) loci. Based on the mean centered log ratio (MCLR) of HpaII/MpaI (Khulan, et al), there were negligible global changes in methylation between the 4 matched pretreatment and relapse samples. Using cut-off values to select for hypermethylated (HR) DNA (MCLR ≤0.0) and more completely HO DNA (MCLR ≥1.0) revealed some decrease in the ratio of HR:HO DNA at relapse in 3 of the 4 patients, primarily due to an increased fraction of HO DNA (see table).

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We then assessed relapse-associated changes in methylation in 3,908 promoters in which the change from pretreatment to relapse was associated with p <0.05 (paired t-tests) across all 4 data sets. A change from HO to HR was defined as a decrease in the MCLR by ≥2-fold in the relapse group; a change from HR to HO was the opposite, i.e., an increase in MCLR by ≥2-fold. There was a change from pretreatment HO to relapse HR for 563 loci (7.5% of effective HO promoters) and from pretreatment HR to relapse HO for 949 promoters (8.6% of effective promoters). More stringently, when promoters were selected for a change in MCLR from pretreatment HO ≥1.0 to relapse HR ≤0.0 or, conversely from pretreatment HR ≤0.0 to relapse HO ≥1.0, 40 and 82 promoters, respectively, were identified with p ≤0.05. These results indicate that changes in gene promoter methylation from pretreatment HR to relapse HO are more common than the reverse, and they are contrary to our pre-study hypothesis that increased global DNA methylation might be a major contributor to molecular progression processes in APL cells leading to clinical relapse.