Aberrant Methylation of Tumor Suppressor Genes in Chronic Lymphocytic Leukemia

Aberrant methylation of CpG islands of the promoter regions of tumor suppressor genes (TSG) is now considered an important epigenetic mechanism for gene inactivation. Chronic lymphocytic leukemia (CLL) is the most frequent type of adult leukemia in the Western world, accounting for about 30% of all leukemic cases. It is characterized by a highly variable clinical course with survival times ranging from months to decades. Some patients have a more stable, indolent disease whereas others exhibit a progressive disease requiring early therapy. During this prolonged period of clinical history, different genetic alterations may be sequentially acquired, including aberrant promoter hypermethylation. In this study, we have evaluated methylation status of p15^INK4b, p16^INK4a, p14^ARF, SOCS-1, p27^KIP1, RASSF1A and TP73 (TAp73 isoform) genes in patients with CLL. Results were correlated with clinicopathologic characteristics of these patients. Thirty-three CLL patients (16 males; median age 65 years; Rai stage: 0: 9, I–II: 14 and III–IV: 10) were evaluated. All patients gave informed consent and the study was approved by the Ethics Committee of our Institution. Peripheral blood samples from 10 normal individuals and CpGenome Universal Methylated DNA (Chemicon International) were used as negative and positive controls, respectively. DNA was extracted from peripheral blood cells from patients and controls using phenol/chloroform method. Methylation status was performed using Methylation Specific PCR technique and DNA sequencing. For statistical analysis, Student’s t and Fisher’s exact tests were used. The methylation index (MI; ratio between the number of genes methylated and the number of genes analyzed) was also calculated. Cytogenetic and FISH (fluorescence in situ hybridization) analysis using specific DNA probes for CLL (LSI p53/ATM/13q14/13q34/CEP12, Vysis-Abbott) were also performed. The gene most frequently methylated was TP73 (70% of cases). Frequencies of p15^INK4band p16^INK4a gene methylation were 9% and 3%, respectively. A coexistence of p15^INK4b and TP73 gene methylation was observed. No patient showed methylation in p14^ARF, SOCS-1, p27^KIP1 and RASSF1A genes. The methylation index ranged from 0 to 0.29 with a median of 0.14, corresponding to one gene/sample. No significant differences in the frequencies of cytogenetic (31,3% and 50%) and FISH genomic alterations (62% and 87,5%) between patients with and without aberrant methylation, respectively, were found. Similarly, the correlation with clinical characteristics: sex, age, Rai stage, lymphocytosis, β₂ microglobulin, LDH, and treatment free survival in both groups of patients did not reach statistical significance. Our findings suggest that aberrant methylation of TP73 gene is a frequent event in this pathology. TAp73 isoform is involved in cell cycle arrest and apoptosis. Thus, its silencing could be important for CLL cells survival. Simultaneously, our data would confirm that, in Caucasian population, the methylation of p15^INK4band p16^INK4a gene promoters can be detected in a small subset of CLL patients.