Epigenetic differences in cytogenetically normal versus abnormal acute myeloid leukemia

Abstract

Background: Methylation of tumor suppression genes (TSGs) is common in myeloid malignancies. However, application of this as a molecular marker for risk stratification in patients with AML is limited. Design and Methods: To elucidate the impact of patterns of TSG methylation on outcome in cytogenetically normal patients, 106 samples from patients with having normal cytogenetic AML were evaluated for methylation of 12 genes by MSP. For sake of comparison, samples from patients with AML and abnormal cytogenetics (n=63) were also evaluated. Results: Methylation frequencies in the whole group (n=169) were similar to previous reports for CDH1 (31%), ER (31%), FHIT (9%), p15^INK4b (44%), p73 (25%), and SOCS1 (75%). Methylation of CTNNA1 was observed in 10%, CEBP-α in16%, CEBP-δ in 2%, MLH1 in 24%, MGMT in 11% and DAPK in 2% of AML samples. We find that DNA methylation was more prevalent in patients with normal compared to karyotypically abnormal AML for most genes; CEBPα (20% vs 9%), CTNNA1 (14% vs 4%), and ER (41% vs 19%) (p<0.05 for all comparisons). In contrast, p73 was more frequently methylated in patients with karyotypic abnormalities (17% vs 38%; p<0.05), perhaps due to specific silencing of the pro-apoptotic promoter shifting p73 gene expression to the anti-apoptotic transcript. In AML patients with normal cytogenetics, TSG methylation was not associated with event free or overall survival in a multivariate analysis. Conclusions: In patients with AML, TSG methylation is more frequent in patients with normal karyotype than those with karyotypic abnormalities but does not confer independent prognostic information for patients with normal cytogenetics.

Acknowledgements

This work was supported by The Flight Attendant Medical Research Institute (FAMRI), grant number 032053. MAM would like to acknowledge the Department of Defense (MPD510343) for support. This work was also supported by the NCI Cancer Center Support Grant (CA06793) which helped to support the Specimen Acquisition Core (SAC) Laboratory for sample collection and storage.

Conflict of Interest

Dr. James Herman is a consultant to and receives research support from OncoMethylome Sciences. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies. All the other authors have no conflicts of interests to disclose.

Figures and Tables

Figure 1 (A) Methylation rates by gene for each of the genes examined across the recognized cytogenetic risk groups. The good risk group includes 10 patients with t(8; 21), inv(16) or t(15:17) mutations, the intermediate group includes 106 patients with normal karyotype leukemia and 14 patients with <3 karyotypic abnormalities, the adverse group includes 30 patients with complex karyotype and 9 patients with abnormalities involving chromosomes 5, 7 or 11q23. No significant differences were observed between each of these cytogenetic risk groups. (B) When patients with normal karyotype were compared to those with a structural karyotype abnormality, differences in methylation frequency were observed. Methylation of CEBP-α, CTNNA1, ER and p73 (*) were significantly different between the two groups (p < 0.05).

Figure 2 (A) No significant differences were observed between rates of methylation for normal karyotype samples with and without a FLT3-ITD mutation. (B) No significant differences in rates of methylation were observed between samples with and without a NPM1 mutation.

Figure 3 (A) No significant differences were seen between samples obtained from patients with de novo leukemia and those presenting with a history of an antecedent hematologic abnormality. Patients in the latter group were older, with a median age of 69 years vs. 52 years in the de novo group, and all had a diagnosis of AML at the time of sample acquisition. (B) There were no differences in methylation between normal karyotype samples obtained at the time of diagnosis as compared to those obtained at the time of AML relapse.

Figure 4 (A) Schematic of p73 gene demonstrating its 14 exons and the location of our MSP and RT-PCR primers. The TAp73 transcript includes exons 1–14 and has a dense CpG island upstream of the transcription start site. The DNp73 transcript includes exons 3–14. (B) Methylation Specific PCR reactions for leukemia cell lines KG1a, U937, HL60 and ML1. KG1a and U937 demonstrate complete methylation, HL60 is hemimethylated for p73, ML1 is unmethylated. NL is a negative control for methylation in normal peripheral blood lymphocytes and IVD is a positive control for methylation. (C) RT-PCR for the long (TAp73) and short (DAp73) transcripts of p73. TAp73 exerts a pro-apoptotic and DNp73 an anti-apoptotic effect. Methylation of the p73 promoter preferentially silences the long transcript of p73 while allowing continued expression of the short transcript, favoring an anti-apoptotic phenotype. (D) Treatment for 72 h with 1 µM 5-azacytidine (5AC) reverses methylation, while untreated (mock) and SAHA treated cell lines maintain a methylated phenotype. (E) Treatment of KG1a and U937 cell lines with 5AC results in re-expression of silenced TAp73 (both) and DNp73 (KG1a).

Figure 5 Uniformly treated patients with normal karyotype AML at initial diagnosis (n = 72) (A) Multivariate hazard ratio for relapse by gene with 95% confidence intervals designated by black bars. (B) Multivariate hazard ratio for death by gene corrected for age, antecedent cytopenia(s) at diagnosis, FLT3-ITD and NPM1 mutational status and total white blood cell count at time of AML diagnosis with 95% confidence intervals designated by black bars.

Table 1 Characteristics of the study population