Increased methylation of the tumor suppressor and all-trans retinoic acid (ATRA) target gene RARβ2 has been reported in a variety of neoplasias. In APL, this has been related to the recruitment of DNA methyltransferases (DNmts) by PML-RARα to the RA response element-containing RARβ2 gene promoter (

Di Croce, et al, Science 295, 1079, 2002
). In this study, we applied a highly quantitative method, pyrosequencing of bisulfite-converted DNA, to precisely measure the level of methylation (% of total dinucleotides;dnts) at each of 14 CpG dnts in the RARβ2 promoter from APL cases with de novo vs 1st relapse vs ≥2nd relapse disease. Additionally, we assessed whether patients with PML-RARα mutations at relapse, which increase binding to the co-repressor complex and might secondarily increase DNmt recruitment to this complex, are associated with increased methylation of this ATRA target gene. Non-parametric 2-sided Wilcoxon tests were utilized to evaluate potential differences in median values. Surprisingly, the methylation value for de novo APL cells (N = 10) was only slightly greater than that for normal peripheral blood mononuclear cells (N=4) (medians of sums of all 14 dnts were 95.9 and 68.9, respectively; p = 0.14). However, in a matched set of pretreatment and 1st relapse samples from 7 patients treated on intergroup protocol INT0129/E2491, the methylation level was significantly higher in the relapse samples at 6/14 dnts (p <0.05 each) and in the sums of all dnts (p = 0.03). Compared to these 7 1st relapse cases, the median values for each of the 14 dnts and the sums of all dnts were higher for 6 cases with ≥2 relapses (median of sums 149.8 vs 385.6, respectively) but this difference was not significant (p = 0.22), primarily related to 1 double-relapse case with exceptionally low values (excluding this case, p = 0.05). Similarly, compared to 8 relapse cases without PML-RARα mutations, 5 relapse cases with mutations had higher median values of 13/14 dnts and of the sums of all dnts (160.5 vs 402.0) but this was not statistically significant (p = 0.27). These results indicate that higher levels of RARβ2 promoter methylation are present in relapse compared to de novo APL cells and suggest that increasing methylation is frequently associated with disease progression and with mutations in PML-RARα.

Topics:
acute promyelocytic leukemia, genes, methylation, tretinoin, progressive multifocal leukoencephalopathy, dna modification methylases, disease progression, dna, pyrosequencing, peripheral blood mononuclear cell

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2005, The American Society of Hematology
2004
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