Epigenetic regulation of microRNAs in acute lymphoblastic leukemia.

PURPOSE: To identify microRNAs (miRNAs) epigenetically regulated in acute lymphoblastic leukemia (ALL). METHODS: We first examined ALL-derived cell lines for the presence of abnormal levels of two different histone modifications (trimethylation of H3 lysine 4 [K4H3me3] and dimethylation of H3 lysine 9 [K9H3me2]) in the 5'UTR regions around CpG islands of 78 miRNAs by chromatin immunoprecipitation (ChIP)-on-ChIP analysis. Methylation status (methylation-specific polymerase chain reaction [PCR]) and expression (quantitative PCR) of miRNAs showing a pattern of histone modifications linked to a closed chromatin structure were analyzed in a panel of six ALL cell lines and in 353 ALL patients. RESULTS: CpG islands around 13 miRNAs disclosed high levels of K9H3me2 and/or low levels of K4H3me3, a pattern of histone modifications underlying a closed chromatin structure associated with repressive gene expression. Complete consistency in the correlation between both histone marks, the presence of DNA methylation around these miRNAs, and their expression patterns was confirmed in the six ALL cell lines. Treatment with 5-Aza-2'-deoxycytidine upregulated the expression levels of these genes, suggesting that epigenetic mechanisms deregulate the expression of these miRNAs. A total of 65% of the ALL samples had at least one miRNA methylated (methylated group). Estimated disease-free survival (DFS) and overall survival (OS) at 14 years were 78% and 71% for nonmethylated patients and 24% and 28% for methylated patients (P = .00001 for both). Multivariate analysis demonstrated that methylation profile was an independent prognostic factor for predicting DFS (P = .0001) and OS (P = .0001). CONCLUSION: Aberrant miRNA methylation is a common phenomenon in ALL that affects the clinical outcome of these patients.

Chimerism status is a useful predictor of relapse after allogeneic stem cell transplantation for acute leukemia.

BACKGROUND AND OBJECTIVES: The role of hematopoietic chimerism after allogeneic stem cell transplantation (SCT) for acute leukemia remains controversial. We studied the relationship between hematopoietic chimerism and several prognostic variables on the outcome of SCT in patients with acute leukemia. DESIGN AND METHODS: Chimerism was determined by a semiquantitative method, based on polymerase chain reaction (PCR) amplification of variable number tandem repeat (VNTR) minisatellites, in 133 consecutive patients who underwent allogeneic SCT for acute leukemia (68 myeloid, 58 lymphoid and 7 biphenotypic), all receiving a myeloablative conditioning regimen. RESULTS: The median follow-up for the surviving patients was 44.8 months (range: 12.0-129.0). Recipient hematopoiesis (mixed chimerism, MC) was detected in 40 cases (30.1%). Two types of patients could be distinguished in this MC group: 29 with increasing MC and 9 with decreasing MC. The remaining 93 cases maintained complete donor chimerism (CC) over the whole follow-up period. Patients with increasing MC showed a significantly higher (p<0.001) rate of relapse (93.1%) and death (89.7%) in comparison to both those with CC (26.9% relapse, 44.1% dead) or decreasing MC (11.1% relapse, 44.4% dead). The detection of increasing MC preceded relapse by a median of 74 days (range: 5-434) and was significantly related with the absence of chronic graft-versus-host disease. Univariate and multivariate analysis confirmed that chimerism was the most significant variable involved in relapse, leukemia-free survival and overall survival after SCT. INTERPRETATION AND CONCLUSIONS: These results demonstrate that sequential determination of chimerism allows the prediction of relapse and death after SCT for acute leukemia. The interval between detection of increasing MC and relapse may permit timely implementation of therapeutic measures.

5' CpG island hypermethylation is associated with transcriptional silencing of the p21(CIP1/WAF1/SDI1) gene and confers poor prognosis in acute lymphoblastic leukemia.

The p21 is a downstream effector of p53/p73 and belongs to the CIP/KIP family of cyclin-dependent kinase inhibitors (CDKIs). It is, therefore, a potential tumor suppressor gene and probably plays an important role in tumor development. Moreover, reduced expression of p21 has been reported to have prognostic value in several human malignancies. In contrast with other CDKIs, mutational inactivation of p21 is infrequent, but gene inactivation by an alternative mechanism seems to be the general pathway. In this study, we analyzed the methylation status of the p21 promoter region using semiquantitative polymerase chain reaction in 124 patients with acute lymphoblastic leukemia (ALL). We observed p21 hypermethylation in bone marrow cells from 41% (51 of 124) of ALL patients. Hypermethylation within promoter strongly correlated with decreased p21 messenger RNA expression in tumoral cells. Clinical, molecular, and laboratory features and complete remission rate did not differ significantly between hypermethylated and normally methylated patients. Estimated disease-free survival (DFS) and overall survival at 7 and 9 years, respectively, were 59% and 65% for healthy patients and 6% and 8% for hypermethylated patients (P =.00001 and P =.006). Multivariate analysis of potential prognostic factors demonstrated that p21 methylation status was an independent prognostic factor in predicting DFS (P =.0001). Our results indicate that the p21 gene is subject to methylation regulation at the transcription level in ALL and seems to be an important factor in predicting the clinical outcome of these patients.