Research Article For reprint orders, please contact: reprints@futuremedicine.com MGMT pyrosequencing-based cut-off methylation level and clinical outcome in patients with glioblastoma multiforme Lorena Gurrieri 1,2 , Elisa De Carlo 1,3 , Lorenzo Gerratana* ,1,4 , Giovanna De Maglio 5 , Marianna Macerelli 1 , Federica Edith Pisa 4 , Elena Masiero 5 , Giuseppe Aprile 1,6 , Alessandro Follador 1 , Fabio Puglisi 3,4 , Gianpiero Fasola 1 , Simona Rizzato 1 & Stefano Pizzolitto 5 1 Department of Oncology, University Hospital of Udine, Udine 33100, Italy 2 Department of Oncology, ASUITS University Hospital, Trieste 34129, Italy 3 Department of Clinical Oncology, CRO Aviano National Cancer Institute, Aviano 33081 (PN), Italy 4 Department of Medicine (DAME), The University of Udine, Udine 33100, Italy 5 Department of Pathology, University Hospital of Udine, Udine 33100, Italy 6 Department of Oncology, San Bortolo General Hospital, ULSS8 Berica, East District, Vicenza 36100, Italy * Author for correspondence: lorena.gurrieri@gmail.com Aim: MGMT promoter methylation has been associated with improved survival in glioblastoma multiforme treated with temozolomide. However, there is no consensus on specifc cut-off levels of methylation. The aims of the study were to explore the prognostic impact of MGMT methylation status and to analyze the role of specifc cut-off values. Materials & methods: We analyzed 108 glioblastoma multiforme pa- tients treated between 2008 and 2013 stratifed according to three pyrosequencing-based quantitative methylation in: unmethylated (methylation <9%), intermediate (9–29%) and highly methylated (>29%). Results: The three-class stratifcation has a prognostic impact (median progression-free survival: 7.97, 11.6 and 15 months respectively; p = 0.004; median OS: 13.2, 15.8 and 19.5 months, respectively; p = 0.0002), especially in patients exposed to temozolomide. Conclusion: Our study confrmed that the independent prognostic role of MGMT methylation status. An average level of methylation between all investigated CpGs of 9% may help discriminating between methylated and unmethylated tumors. First draft submitted: 31 August 2017; Accepted for publication: 7 December 2017; Published online: 9 March 2018 Keywords: CpG islands • cut-off • glioblastoma • IDH1 • methylation • MGMT • pyrosequencing • temozolomide Glioblastoma multiforme (GBM), a highly aggressive disease characterized by infiltrating growth, is the most common malignant primary brain tumor and it represents 50% of gliomas and 20% of all cancers [1]. Despite the advances in multidisciplinary treatment, the prognosis remains poor [2], with a median survival ranging from 9 to 22 months [3]. Surgical resection, followed by radiotherapy and sequential temozolomide (TMZ), also known as ‘Stupp protocol’, is the current therapeutic gold standard [4]. The MGMT gene is located in the 10q26 locus and encodes for an enzyme involved in the DNA repair system that acts by removing alkyl groups from the O-6 position of guanine [5]. MGMT expression is regulated through the hypermethylation of the CpG islands within both the promoter and enhancer regions of the gene [6]. This phenomenon, called epigenetic silencing, represents a resistance mechanism against alkylating agents [7] and it is a proved prognostic biomarker in GBM patients [7–11]. Retrospective studies have consistently shown that MGMT methylation may enhance the response to alkylating- based regimens, such as TMZ [9–11], also among elderly GB patients [12–14]. Because of the increasing clinical evidence, the implementation of diagnostic tests assessing MGMT methylation is rapidly growing. However, the best approach to adopt is widely debated. PCR-based techniques, such as methylation-specific PCR (MSP), are the most common methods of assessment [8], but DNA sequencing (i.e., bisulfite genomic sequencing) provides a more complete information on methylation status [15]. Future Oncol. (Epub ahead of print) ISSN 1479-6694 10.2217/fon-2017-0437 C  2018 Future Medicine Ltd