Methylation-specific PCR demonstrates preferential promoter methylation of genes of interest in MLL-r ALL compared with MLL-wt ALL and normal controls. DNA from the REH cell line and HELP primary samples were treated with bisulfite to specifically convert unmethylated cytosines to uracil. PCR primers for anticipated post–bisulfite-methylated (M) and unmethylated (U) promoter sequences for the genes DAPK1, p73, CCR6, and HRK were used to determine their methylation status. (A) REH cells were used as control to assure complete bisulfite conversion because in REH cells the DAPK1 promoter is completely unmethylated whereas the p73 promoter is completely methylated. (B) Examples are shown from treatment of the HELP assay primary samples. In DAPK1 and HRK, only the MLL-r sample shows a methylated amplicon. In CCR6, although all samples show methylated amplicons, the MLL-r sample is clearly preferentially methylated as it shows only a dim unmethylated amplicon. (C) Densitometry was performed on all amplicons for all primary samples in each group and average unmethylated amplicon–methylated amplicon (U:M) densitometry ratios and their SDs are shown. The DAPK1 and HRK promoters are preferentially methylated in MLL-r ALL cells in comparison with other leukemias and normals; CCR6 promoters have some methylation in all leukemias, whereas in MLL-r ALL it is preferentially methylated and preferentially unmethylated in normals. (D) With the exception of, in the CCR6 gene, MLL-r versus other leukemias and MLL-r versus hyperdiploid ALL (where hyperdiploid leukemia showed greater methylation than MLL-r) all genes and groups compared trended toward statistical significance, with MLL-r leukemia having a lower U:M ratio (more methylation); despite small numbers, several comparisons reached statistical significance and are listed.