MPTH-14PROGNOSTIC ROLE OF METHYLATION STATUS OF MGMT PROMOTER ON GLIOBLASTOMA PATIENTS ESTIMATED QUANTITATIVELY BY PYROSEQUENCING

Abstract

OBJECTIVE: This study was aimed to determine whether pyrosequencing might be useful to determine methylation status of MGMT promoter using relatively old archival tissue samples of glioblastomas as a clinical biomarker. Additionally, we examined other prognostic factors for survival of glioblastoma patients. METHODS: The available study set included FFPE tissue from 104 patients at our two hospitals from 1997 to 2012, of which all were diagnosed as glioblastoma. With review of medical records, clinicopathological data were collected. Deoxyribonucleic acid (DNA) extraction and bisulfite conversion was performed from three (10 µm thickness) slices of FFPE material using the QIAamp DNA FFPE extraction kit and EpiTect bisulfite kit (Qiagen, Hilden, Germany), respectively. RESULTS: Among them, methylated MGMT promoter was found in 43 (41.3%) of samples. The average percentage of methylation was 14.0 ± 16.8%, and methylated cases were 39.0 ± 14.7%; that of each year did not show a linear increasing or decreasing pattern according to the age of the FFPE block (p = 0.687). In the multivariate analysis, age (p = 0.048), performance status (p = 0.002), extent of surgery (p < 0.05), method of adjuvant therapy (p < 0.05), and methylation status estimated by MSP (p = 0.034) and pyrosequencing (p = 0.001) were independently associated with overall survival. Additionally, patients with more methylated cases (≥ 39%) had longer survival than those with less methylated cases (≥ 9% to < 39%) (HR of 7.529, 95% CI 3.956-11.102; p = 0.016). CONCLUSION: In this study, status and extent of methylation in MGMT gene promoters analyzed by pyrosequencing were found to be associated with overall survival in glioblastoma patients. Pyrosequencing is a quantitative method overcoming the problems of MSP, and is a simple technique for accurate analysis of DNA sequences.