Aberrant DNA Methylation of a Cell Cycle Regulatory Pathway Composed of p73, p15 and P57kip2 Is Associated with Poor Prognosis in Adult Patients with Philadelphia Chromosome Negative Acute Lymphocytic Leukemia (ALL).

Abstract

Aberrant DNA methylation of promoter-associated CpG islands is a frequent event in ALL. The high frequency of aberrant DNA methylation observed in patients with ALL, and the fact that these epigenetic changes are also present at relapse suggest that aberrant DNA methylation has a role in the pathogenesis of the disease. The identification of subgroups of patients with poor prognosis based on their methylation characteristics may translate in specific clinical interventions, such as the introduction of early allogeneic bone marrow transplantation or the use of hypomethylating agents. In initial studies, we have shown that methylation of more than 1 gene of a cell cycle regulatory pathway composed of p73, p15 and p57KIP2 occurs in close to 25% of patients, and is associated with a poor prognosis in patients with Philadelphia chromosome negative (Ph negative) disease (Blood 2003; 101:4131). Of importance, protein expression of this pathway was associated with a significant better prognosis by multivariate analysis (JCO 2005; 23:3932–9). To confirm the prognostic value of methylation of p73, p15 and p57KIP2, we are conducting an NCI-sponsored large-scale study of 300 patients wit Ph negative ALL at the time of initial presentation. As part of this study, and based on the original statistical design, we have performed an interim analysis of the first 93 patients studied. The objective of this interim analysis was to terminate this study early in case of a high likelihood that the study will be negative: that is that methylation of triad of genes is not associated with a poor prognosis. To detect DNA methylation, we have developed a new real-time bisulfite PCR assay. This method allows for the simultaneous analysis of multiple samples in less than 24 hours, is quantitative, and requires less than 0.2 ug of DNA for each gene. To amplify bisulfite treated DNA, we designed primer sets and probes for all three genes in regions known to be inversely correlated with gene expression. To quantify methylation density, we used the interferon gamma (INFG) gene as an internal control because it has very rare CpG sites, is a single copy gene and has no homology with other known genes. The characteristics of the patients are similar to those of the initially reported cohort of patients (Blood 2003; 101:4131–6). The median overall survival (OS) of the whole group was 166 weeks. Out of the 93 patients, 88 (94%) achieved a complete remission. And out of these 88 patients, 37 (42%) eventually relapsed. The median disease-free survival was 127 weeks. P57KIP2 was methylated in 24% of patients, p15 in 34% and p73 in 21%. Methylation of more than 1 gene of this triad was observed in 19% of patients. This subgroup of patients had a median OS of 50 weeks. By multivariate analysis, methylation of more than 1 gene of this pathway (hazard ratio 4.07, 95% CI 1.63–7.5, p=0.003), presence of the CALLA antigen, older age, increased WBC and lower platelet count were associated with a significantly worse prognosis. Based on this interim analysis, this study continues, and will be completed by the time of the meeting. These results indicate that analysis of methylation markers may have a role in the prognostication of patients with Ph negative ALL.