A LARGE, MULTICENTER, RETROSPECTIVE STUDY TO IDENTIFY A CUT-OFF OF MGMT METHYLATION STATUS BY QUANTITATIVE PYROSEQUENCING APPROACH IN PATIENTS (PTS) WITH GLIOBLASTOMA (GBM). Background: MGMT promoter methylation status representsan important prognostic factor for GBM PTS in terms of progression free survival (PFS) and overall survival (OS). Quantitative pyrosequencing approach is a valid alternative to methylation-specific PCR but a cut-off value in still unclear. We performed a large, multicenter, retrospective study to identify a real cut-off value to discriminate its impact on clinical outcome in terms of PFS and OS. Methods: Retrospectively, from italian neuro-oncology centers, we collected GBM PTS from 2005 to 2016 with assessment of MGMT promoter methylation by pyrosequencing approach evaluating CpG islands from 74 to 83. Other inclusion criteria were: confirmed histological diagnoses of GBM, ECOG PS ≥2, treatment with concomitant radiation therapy and temozolomide. Kaplan-Maier method was used to estimate the survival curves and ROC curve for defining cut-off value for PFS and OS. Results: 376 GBM PTS were enrolled;median age was 62 ys (25-86); ECOG PS was 0 in 129 PTS, 1 in 160 PTS, 2 in 87 PTS; 212 PTS (58%) had a complete resection. 67 PTS (18%) received a second surgery. Median PFS and OS was 8.6 and 14.3 months. The optimal cut-off value to identify a strong prognostic value of MGMT methylation status in terms of PFS and OS, was 26% (sensitivity 72%, specificity 61%, accuracy 71%) and 24% of methylation (sensitivity 72%, specificity 61%, accuracy 71%), respectively. On multivariate analyses, corrected for age, KPS, type of surgery and second surgery, the MGMT cut-off values remained significantly correlated to longer PFS (HR = 0.5, 95%CI 0.4-0.7) and OS (HR = 0.47, 95% CI 0.3-0.6). Conclusions: From this large, multicenter study, we identify, by pyrosequencing approach, a strong prognostic value of MGMT methylation, in terms of PFS and OS. This value could be used as stratification factor in prospective clinical trials.