Extensive intra- and interindividual heterogeneity of p15(INK4B) methylation in acute myeloid leukemia

Silencing of the cyclin-dependent kinase inhibitor gene p15(INK4B) by cytos ine methylation of the promoter region has been associated with some types of hematological malignancy. To study in detail the patterns of p15(INK4B) methylation in patients with acute myeloid Leukemia, lie adopted a novel ap proach based on PCR amplification of bisulfite-treated DNA followed by reso lution of differentially methylated sequences by denaturing gradient gel el ectrophoresis. This method visually displays the degree and heterogeneity o f DNA methylation and enables the isolation and characterization of distinc t clonotypic epigenotypes. A surprisingly high degree of intra- and interin dividual heterogeneity of p15(INK4B) methylation mas observed in the 65 acu te myeloid leukemia patients examined, Methylation was detected in 46 (71%) of the patients and mas observed more frequently in the French-American-Br itish subtypes M1/M2 than in M4/M5 (P < 0.025). Examination of the same pan el of samples using a highly sensitive methylation-specific PCR method shel ved methylated p15(INK4B) alleles in 61 (94%) of the samples. We present ev idence that the higher frequency of p15(INK4B) methylation determined by me thylation-specific PCR may, at least in part, be due to the presence of a s malt fraction of p15(INK4B)-methylated lymphocytes in normal blood.