Frequency of aberrant promoter methylation of p15INK4b and O6-methylguanine-DNA methyltransferase genes in b-cell non-Hodgkin lymphoma: A pilot study

The methylation status of the target promoter sequences of p15INK4B (p15) and O6-methylguanine-DNA methyltransferase (MGMT) genes was studied by methylation-specific PCR in 10 adult patients with de novo B-cell non- Hodgkin lymphoma (B-NHL). The aberrant hypermethylation of the p15 gene was more frequent (50%) compared to the hypermethylation of the MGMT gene (30%), and was detected in different types of B-NHL in both genes. Hypermethylation of the MGMT gene occurred exclusively in association with the hypermethylation of the p15 gene. All lymphoma patients with hypermethylation of the p15 and/or MGMT genes had a higher clinical stage of the disease (IV - V). We show the association of anemia and/or thrombocytopenia with the hypermethylation of the p15 gene, ascribing the p15 gene as a potential prognostic marker in B-NHL. Comethylation of MGMT with the p15 gene represents a novel finding and presents both genes as candidates for future studies of the hypermethylation profiles of B-NHL.

Homozygous Deletion of p16, p14 and p15 Is a Poor Prognostic Factor in Adult B-Lineage Acute Lymphoblastic Leukemia, Not in Childhood B-ALL: A Comparison through Deletion and Hypermethylation Study

Backgrounds: The biologic characteristics of childhood acute lymphoblastic leukemia (ALL) is different from those of adult ALL. Tumor suppressor genes, p16, p14, and p15 gene are inactivated either by promoter methylation, deletion or mutation, however, in leukemia, promoter methylation and deletion are the main mechanisms of inactivation. Aims: To compare the alteration status of p16, p14, and p15 gene in childhood and adult ALL, we analyzed the incidences and the prognostic significances of deletion and hypermethylation of p16, p14, and p15 in childhood and adult B-ALL. The association between alterations of those genes and known cytogenetic prognostic factors (BCR-ABL, TEL-AML, MLL rearrangement, and numerical changes) were also assessed. Methods: A total of 91 newly diagnosed B-ALL patients (61 children, 30 adults) were studied. Interphase fluorescent in situ hybridization study (p16, BCR-ABL, TEL-AML, MLL) and methylation specific PCR were performed using bone marrow mononuclear cells. Numerical changes were assessed by FISH and chromosome analysis. Chi-square test, Fisher’s exact test, Kaplan and Meier method and Cox proportional hazards regression were applied for statistical analysis. Results: The frequencies of homozygous deletion of p16, p14, and p15 were 11.5% in children and 30.0% in adult, showing higher incidence in adults (p=0.029). In overall survival study, homozygous deletion was associated with the worse prognosis in adults (Fig 1, p=0.019), but not in childhood. The incidences of promoter methylation of p16, p14, and p15 were as follows: 34.4%, 14.8%, and 34.4% in children; 26.7%, 10.0%, and 40.0% 26.7% in adults, respectively, with no statistical difference between two groups. No significant association was observed between deletion and hypermethylation. Childhood ALL showed inactivation of p16 (39.3%), p14 (24.6%), and p15 (42.6%), while adult ALL showed inactivation of p16 (46.7%), p14 (33.3%), and p15 (56.7%), with the same order of frequencies, but with higher tendency of methylation in adult ALL. In p14 unmethylated adults, the homozygous deletion had adverse effect on overall survival (OS) (p=0.036). There were no significant association between chromosomal aberrations and promoter methylation in childhood and adult ALL. The children with sole MLL rearrangement showed poorer disease free survival (DFS) than those with sole homozygous deletion with low statistical significance (p=0.059). Homozygous deletion was translated into poor prognosis in OS in adults without MLL rearrangement (p=0.011). Adult with normal karyotype showed shorter OS when accompanied by homozygous deletion, although p value was 0.051. Conclusions: We performed a comprehensive analysis of deletion and hypermethylation of p16, p14, and p15 genes in both childhood and adult B-ALL. Homozygous deletion was more frequent in adults, showing association with shorter OS in adults, but not in children. This difference of distribution and prognostic value between childhood and adult ALL could be one of the explanations for the disparity of clinical outcome. Our results suggest that homozygous deletion is an independent prognostic factor in adult ALL. Table 1. Deletion and methylation profiles of p16, p14, and p15, and their prognostic siginificances P16, P14, and P15 Deletion P16 Methylation P14 Methylation P15 Methylation *P: p value by multivariate analysis †OS: Overall survival ‡DPS: Disease free survival Childhood Frequency 11.5% 34.4% 14.8% 34.4% *P (†OS) 0.853 0.979 0.651 0.591 P (‡DPS) 0.716 0.956 0.809 0.977 Adults Frequency 30.0% 26.7% 10.0% 40.0% P (OS) 0.019 0.151 0 892 0.330 P (DPS) 0.218 0.382 0.079 0.760 Figure 1. Kaplan-Meier curve for childhood and adult B-ALL patients.
 P value was obtained by multivariate analysis using Cox hazard regression model. (A) Childhood B-ALL (B) B. Adult B-ALL Figure 1. Kaplan-Meier curve for childhood and adult B-ALL patients.
 P value was obtained by multivariate analysis using Cox hazard regression model. (A) Childhood B-ALL (B) B. Adult B-ALL

Methylation Profiles of p16, p15 and p14 Genes in Korean Patients with Multiple Myeloma.

Dysruption of cell cycle control genes (p16, p15 and p14) is known to be involved in the tumorigenesis of multiple myeloma (MM). We investigated the inactivation status of p16, p15 and p14 genes using promoter methylation study and fluorescent in situ hybridization (FISH) study in patients with MM and analyzed the association of inactivation of those genes and clinical prognosis.MM Promoter methylation study of p16, p14 and p15 gene was done with newly diagnosed 52 patients with by bisulfite modification and methylation specific PCR. Two sets of primers were used for p16 methylation study and one set of primer was used for p15, p14 methylation study. Deletion of p16, p14 and p15 gene was detected by dual color FISH (Vysis, Downers GroveIL, USA). Overall survival was analyzed by Kaplan Meier Method and Cox’s proportional hazard model. Methylation of p16, p15 and p14 promotor was detected in 36/52 (69.2%), 15/52(28.8%) and 7/52 (13.5%) patients with MM, respectively. Methylation of any of p16, p15 or p14 promotor was observed in 44/52 (84.6%) of MM patients Deletion of p16, p15 and p14 was detected in only one of the patients with MM. Of note, in the cases (15/52, 28.8%) showing that in cases with two positive methylation specific PCR of the p16 promotor with two sets of primers, significant lower overall survival rate (p<0.036) were observed compared with only one positive methylation specific PCR. Heavy methylation of p16 protomor was a independent predictive variable for overall survival [Hazard Ratio, 3.74; 95% CI, 1.44–9.68, P = 0.007]. Other adverse prognostic factors by univariate analysis were old age (p=0.014), β2-microglobuline (p=0.026), high serum creatinine levels (≥2.0 mg/dL, p=0.014). More than 80% of MM patients showed the methylation of any of p16, p15 or p14 promotor, which suggest the potential applicability of hypomethlyating agents to MM. The promoter methylation of p16, p14 or p15 was a major contributor to the disruption of cell cycle regulation in MM, whereas both the deletion and/or the promoter methylation of p16, p14, and p15 contributed to the disruption of cell cycle regulation in ALL. In our study, methylation of two positive methylation specific PCR of the p16 promotor was an independent adverse prognostic factor in MM. We infer that quantitative methylation study for p16 promotor is helpful for the evaluation of prognosis. Figure 1. Overall survival analysis of patients with according to p16 methylation amount (P = 0.036) Figure 1. Overall survival analysis of patients with according to p16 methylation amount (P = 0.036)