Detection of Residual p73 DNA Methylation Predicts for Shorter Disease Free and Overall Survival in Patients (pts) with Philadelphia (Ph) Chromosome Negative Acute Lymphocytic Leukemia (ALL) in Remission.
Authors: Garcia-Manero Guillermo , Bueso-Ramos Carlos , Xiao Lianchun , Hoshino Koyu , Rosner Gary , Pierce Sherry , Yang Hui 
Published: October 2019

Abstract
Aberrant DNA methylation of multiple promoter CpG islands is a common feature of ALL. Specific DNA methylation patterns involving a triad of hierarchical clustered cell cycle controlling genes including p73, p15 and p57KIP2 predict for poor prognosis in Ph negative ALL (Garcia-Manero, Blood, 2003). Of importance, this aberrant DNA methylation patterns are stable at the time of relapse in a majority of patients. This indicates that aberrant DNA methylation is a fundamental molecular feature of the ALL blast cell (Garcia-Manero, Clinical Cancer Research, 2002). Based on this, we hypothesized that the detection of residual DNA methylation in patients with standard risk (Ph negative) ALL could predict for shorter disease free survival, and worse overall survival (OS) in this patient population. To test this hypothesis, we have analyzed the frequency of p73, p15 and p57KIP2 DNA methylation at the time of initial documentation of complete remission (CR) (days 14 to 21) in 199 patients in CR with Ph negative ALL. All patients had been treated with hyperCVAD-based chemotherapy at our institution. To perform this analysis, we have developed a sensitive and specific novel real-time methylation specific PCR assay to detect DNA methylation. This method requires bisulfite DNA conversion. It uses the interferon-gamma gene as a DNA loading control and can be performed in less than 24 hours. The characteristics of the 199 patients evaluated are: median age 38 years (range 15–83), cytogenetics: 32% diploid, 5% hyper or hypodiploid, 20% insufficient metaphases and 3% MLL+, the rest others. 16% were of T cell phenotype. Using this assay, p73 methylation was detected in 18 (10%) patients, p15 in 33 (16%) and p57KIP2 in 7 (3.5%). Methylation of at least 1 gene of this triad was observed in 48 (24%) patients. The median OS of the whole group was 209 weeks (95% CI 158–514) and the disease free survival (DFS) 146 weeks (95% CI 110–335). These were representative of the overall experience with hyperCVAD at MDACC. By multivariate analysis, the presence of residual p73 methylation on days 14 to 21 in patients with Ph negative ALL was associated with a shorter DFS (hazard ratio 2.68, p=0.003) and a worse OS (hazard ratio 2.69, p=0.002). This was independent of any other patient characteristic. Other characteristic included in these models included age and platelet count for OS, and age, b2-microglobulin, LDH and platelets for DFS. The presence of p15, p57KIP2 methylation or the combination of multiple genes was not associated with shorter OS or DFS. In summary, the detection of residual p73 methylation allows the identification of a subset of patients with ALL in remission and worse prognosis. This data may allow the development of specific therapeutic interventions, such as the use of hypomethylating agents, or early allogeneic stem cell transplantation for this at-risk patient population, and suggest that inactivation of p73 is a key event in ALL.