PF543 PROMOTER METHYLATION OF TUMOR SUPPRESSOR GENES ON THE SHORT ARM OF CHROMOSOME 1 IN MYELODYSPLASTIC SYNDROME AND ACUTE MYELOID LEUKEMIA

Background: We previously reported frequent loss of heterozygosity on the short arm of chromosome 1 (1p) in the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML) as well as chronic myeloid leukemia (CML) to blast crisis. Methylation in a promoter CpG of several tumor suppressor genes has been associated with loss or decreased expression in tumors. We previously reported methylation of the PRDM2 gene in MDS. We also found that the PRDM2, RUNX3, and TP73 genes on 1p36 were frequently methylated in CML. Aims: To elucidate the relevance of tumor suppressor genes on 1p, we extended analysis of promoter methylation and expression of the PRDM2, RUNX3 , and TP73 genes in MDS and AML. Methods: Mononuclear cells were isolated from bone marrow or peripheral blood samples after obtaining written informed consent from 34 patients with MDS, 17 patients with secondary AML from MDS, and 55 patients with de novo AML. The 34 MDS samples consisted of 13 RA, 1 RARS, 10 RAEB, 6 RAEB‐t, 4 CMML and the 55 AML consisted of 1 M0, 12 M1, 17 M2, 7 M3, 8 M4, 7 M5, 1 M6, and 2 M7 (FAB classification). This study was performed according to ethical criteria of our institute. Genomic DNA was treated with bisulfite, and treated DNA was subject to amplification with HotStarTaq Master Mix Kit for MS‐PCR. Bisulfite sequence was performed in both directions. Quantitative real time reverse transcriptase‐PCR was performed in 26 patients of which RNA was suitable for analysis. HL‐60 myeloid leukemia cells were grown in RPMI 1640 in the presence of 5‐Aza‐dC. The correlation between the frequency of methylation and type of disease or clinical characteristics was analyzed using the chi‐square test or Fisher's exact probability test. Analysis was performed using SPSS Software. Results: PRDM2 methylation was detected in 17/34 MDS (50%) and 22/72 AML patients (31%) (p = 0.053). It was detected in 11/17 secondary AML (65%), and 11/55 de novo AML patients (20%) (p = 0.0005). Methylation of the RUNX3 gene was detected in 0/14 MDS, 0/7 secondary AML, and 2/30 de novo AML patients (7%). Methylation of the TP73 gene was observed in 1/14 MDS (7%), 0/7 secondary AML, and 4/32 de novo AML patients (13%). Bisulfite sequence revealed methylation at many CpG sites in the promoter of the PRDM2 gene. PRDM2 expression (mean) was not significantly different in secondary AML and de novo AML (2.026 vs. 1.900, p = 0.815). PRDM2 expression (mean) was not significantly different in methylation‐positive group and methylation‐negative group (1.996 vs. 1.810, p = 0.728). In comparison with expression of normal bone marrow cells, decreased PRDM2 expression was accompanied by methylation in 6/9 patients examined. Decreased PRDM2 expression was also observed in 7/13 patients without methylation. Decreased RUNX3 expression was observed in 5/5 MDS, 5/5 secondary AML, and 12/14 de novo AML patients. Decreased TP73 expression was observed in 2/4 MDS, 2/4 secondary AML, and 6/13 de novo AML patients. 5‐Aza‐dC treatment of HL‐60 cells (that have methylation in PRDM2 ) induced growth suppression, demethylation, and PRDM2 expression. Summary/Conclusion: Methylation of the PRDM2 gene was more frequent than that of the RUNX3 and TP73 genes in MDS and AML. Decreased expression of the genes is caused in part by methylation, whereas another mechanism should be involved in the patients without methylation.