A Novel Method of Microarray Quantitave Analysis of P16 CPG Island Methylation in Leukemia Cells from Patients with Hematological Malignancies.

p16 is a tumor suppressor gene frequently affected by hypermethylation of promoter CpG island in malignant disorders. Phenylhexyl isothiocyanate (PHI) has been shown to induce p16 hypomethylation in leukemia cells. However, the methylation status is usually studied qualitatively or semi-quantitatively at the best. In this study, we developed a novel method to quantify p16 methylation in clinical specimen from patients with hematological malignancies as well as in U266, a multiple myeloma cell line. To establish a standard curve of p16 promoter methylation, methylated and demethylated PCR products were subcloned into plasmids respectively. The clones were selected out as completely methylated and demethylated clones, respectively. The PCR products of the two clones were mixed at different ratios and analyzed on a mciroarray chip to produce a standard curve of methylation. p16 promoter methylation was determined in U266 cells. The baseline p16 methylation was 78.2% in U266 cells. The methylation was decreased to 61.7% and 54.8%, respectively in the presence of PHI of 5 and 10 uM, respectively. We further analyzed p16 methylation status in 7 patients’ specimen, two from blood, 5 from bone marrow. Two patients had ALL, two had AML, one CLL, one MM, and one CMML. 3 patients have p16 gene hypermethylation at 17.7%, 47.5% and 84.3%, respectively. Hypomethylation occurred in two of the three patients’ cells after PHI treatment in vitro (84.3% to 64.8%, and 47.5% to 25.4%). In conclusion, a quantitative microarray method was established for quantitation of p16 methylation status. This will be used for laboratory screening of hypomethylation drugs, such as PHI, and for clinical application to monitor therapeutic efficacy of hypomethylating drugs, such as azacitidine, and to prognostify patients based on p16 methylation level.