Hypermethylation Status of Cancer-Associated Genes in Pediatric Acute Lymphoblastic Leukemia (ALL): Incidence and Potential Implications in Therapy.

Acute Lymphoblastic Leukemia (ALL) is the most common malignancy diagnosed in children, representing nearly one third of all pediatric cancers. Aberrant methylation of CpG island of the promoter region of genes causes gene silencing and could be critical in the initiation and progression ALL. Patients and

The study included a group of 10 de novo pediatric cases of ALL and 18 healthy control children. The cases were treated according to the University Children Hospital protocol and followed-up for 28 days. In the present study we assessed the methylation frequency of p14^ARF, p15^CDKN2B, p16^CDKN2A, MLH1, CTNNB1, and APAF1 genes before cases started therapy (Day 0) and after completing the induction phase at Day 28. DNA was extracted from peripheral blood cells from cases and controls. Methylation status was performed using the MethyLight technique. The methylation index (MI; ratio between the number of genes methylated and the number of genes analyzed) was calculated and results were correlated to minimal residual disease (MRD) status of the cases.

The gene most frequently methylated was p15^CDKN2B (90% of cases on Day 0). Frequencies of p16^CDKN2A, MLH1, CTNNB1 gene methylation were 80%, 70%, and 10% respectively. A coexistence of p15^CDKN2B, p16^CDKN2A and MLH1 gene methylation was observed. No patient showed methylation in p14^ARF and APAF1 genes. The methylation index ranged from 0 to 0.67 with a median of 0.5. After induction therapy was completed (Day 28) the most frequently methylated gene was p15^CDKN2B (80% of cases on Day 28). Frequencies of MLH1, p16^CDKN2A, p14^ARF and CTNNB1 gene methylation were 40%, 30%, 20% and 10% respectively. No patient showed methylation of the APAF1 genes on Day 28. The methylation index ranged from 0 to 0.67 with a median of 0.17 on Day 28. We found that after the induction treatment methylation of p15^CDKN2B, p16^CDKN2A, and MLH1 is a frequent event. Interestingly methylation of p14^ARF was detected after induction. No significant differences in the frequencies of gene methylation and presence of minimal residual disease between patients with and without aberrant methylation, respectively, were found. We found that the methylation status was not associated with patient age at diagnosis, sex, FAB classification and cytogenetic changes.

Our findings suggest that aberrant methylation of p15^CDKN2B gene is a frequent event in this pathology. The relationship between methylation of p15^CDKN2B and leukemia has been reported previously. It appears that methylation of p15^CDKN2B is an early event in ALL and continue to be present even after patients completed the induction treatment. Simultaneously, our data would confirm that, in our cohort, the methylation of p16^CDKN2A and MLH1 gene promoters is a frequent event in pediatric ALL. However, to assess the impact of promoter methylation of these tumor suppressor genes on disease prognosis longer follow-up and a larger patient population is warranted.

No relevant conflicts of interest to declare.