title: MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response.
abstract: BACKGROUND
  The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value.
  
  
  RESULTS
  We analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan-Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP.
  
  
  CONCLUSIONS
  Compared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.
authors.affiliation: Department of Toxicology, University Medical Center of the Johannes Gutenberg University Mainz, Obere Zahlbacher Strasse 67, 55131 Mainz, Germany.
authors.forename: Olivier J
authors.initials: OJ
authors.lastname: Switzeny
authors.affiliation: Department of Toxicology, University Medical Center of the Johannes Gutenberg University Mainz, Obere Zahlbacher Strasse 67, 55131 Mainz, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Christmann
authors.affiliation: Department of Neurosurgery, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstraße 1, 55131 Mainz, Germany.
authors.forename: Mirjam
authors.initials: M
authors.lastname: Renovanz
authors.affiliation: Department of Neurosurgery, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstraße 1, 55131 Mainz, Germany.
authors.forename: Alf
authors.initials: A
authors.lastname: Giese
authors.affiliation: Department of Neuropathology, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstraße 1, 55131 Mainz, Germany.
authors.forename: Clemens
authors.initials: C
authors.lastname: Sommer
authors.affiliation: Department of Toxicology, University Medical Center of the Johannes Gutenberg University Mainz, Obere Zahlbacher Strasse 67, 55131 Mainz, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Kaina
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics*; D005260:Female; D005909:Glioblastoma / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D005910:Glioma / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*; D016019:Survival Analysis; D016896:Treatment Outcome; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D025521:Tumor Suppressor Proteins; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes
keywords: Alkyltransferase; Brain tumors; Epigenetic silencing; Glioblastoma; IDH1; MGMT; Promoter methylation; Temozolomide