Differences in Promoter Methylation of Tumor Suppressor Genes in Cytogenetically Normal and Abnormal Acute Myeloid Leukemias.

Methylation of CpG islands within the promoter regions of a number of tumor suppressor genes (TSG), including CTNNA1, CEBP-α, CEBP-δ, DAPK1, CDH1, ER1, FHIT, MLH1, MGMT, p15^INK4b, p73, and SOCS1 have been reported in patients with myeloid malignancy. Promoter methylation of these genes has been associated with clinical prognosis. In myelodysplastic syndrome (MDS), methylation of CDH1, ER1, FHIT and p15^INK4b have been reported to correlate with higher international prognostic system score (IPSS) and advanced disease. Despite these reports, there has been limited investigation of TSG methylation within the recognized paradigm of risk stratification for acute myeloid leukemias (AML). To elucidate whether distinct patterns of TSG methylation occur within prognostically important cytogenetic risk groups in patients with AML, we obtained 189 bone marrow samples from the Johns Hopkins Hospital leukemia tumor bank. Samples were annotated for age, karyotype, history of antecedent cytopenia(s) or MDS, and treatment. Patients had a median age of 61 years (interquartile range 48–70) and were treated with high dose induction therapy including timed sequential therapy at the Johns Hopkins Hospital. Samples were also analyzed for FLT3-ITD and NPM1 mutation status. For the purpose of analysis, samples were divided into those with normal cytogenetics (n=114) and those with cytogenetic abnormalities [favorable (n=14), intermediate (n=16), single adverse (n=14) and complex (n=30)]. Patients with normal cytogenetics (n=73) who were treated with high dose induction therapy at diagnosis were annotated for overall and event free survival and further subdivided into those with (n=33) and without (n=40) an antecedent hematologic diagnosis, in order to determine if TSG methylation was associated with survival. In addition, we studied patients with normal cytogenetics for whom only relapse samples were available (n=41) to confirm previous reports suggesting that promoter methylation may be enhanced at the time of relapse.

RESULTS: Overall methylation frequencies were similar to those in the reported literature for CDH1 (28%;53/189), ER1 (27%;51/189), FHIT (10%;18/189), p15^INK4b (42%;80/189), p73 (24%;45/189), and SOCS1 (71%;134/189). Methylation of DAPK was infrequent (2%;2/109). Methylation of CTNNA1, CEBP-α, CEBP-δ, MLH1 and MGMT have rarely been reported in patients with AML and were present in 10% (19/189), 15% (28/189), 2% (4/189), 22% (42/189) and 10% (18/189) of samples, respectively. No distinct patterns of methylation were observed amongst the cytogenetic groups. Significant differences in promoter methylation were seen between samples with a normal karyotype and those with any cytogenetic abnormality. CTNNA1 (14 vs. 4%), CEBP-α (24 vs. 9%), ER1 (43 vs. 14%) and FHIT (13 vs. 4%) were more frequently methylated in samples with a normal karyotype, while p73 methylation (12 vs. 35%) was more common in samples with a genetic abnormality (p<0.04 for all comparisons). In patients with normal cytogenetics, methylation of the above genes was not associated with event free or overall survival, even when adjusted for FLT3-ITD and NPM1 mutational status. Furthermore, no differences in methylation frequency were observed for those with and without a history of MDS. In those with a normal karyotype, methylation frequencies for the above genes were similar at diagnosis and relapse. In conclusion, in patients with AML (a) methylation of a number of genes is more frequent in patients with a normal karyotype; (b) in patients with normal cytogenetics, an antecedent hematological diagnosis does not affect methylation frequency; (c) the association of p73 methylation with cytogenetic abnormalities may reflect the known role of p73 in maintaining genetic stability; and (d) no significant acquisition of methylation is observed at the time of relapse in patients with normal cytogenetics.