title: Prognostic value of three different methods of MGMT promoter methylation analysis in a prospective trial on newly diagnosed glioblastoma.
abstract: Hypermethylation in the promoter region of the MGMT gene encoding the DNA repair protein O(6)-methylguanine-DNA methyltransferase is among the most important prognostic factors for patients with glioblastoma and predicts response to treatment with alkylating agents like temozolomide. Hence, the MGMT status is widely determined in most clinical trials and frequently requested in routine diagnostics of glioblastoma. Since various different techniques are available for MGMT promoter methylation analysis, a generally accepted consensus as to the most suitable diagnostic method remains an unmet need. Here, we assessed methylation-specific polymerase chain reaction (MSP) as a qualitative and semi-quantitative method, pyrosequencing (PSQ) as a quantitative method, and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) as a semi-quantitative method in a series of 35 formalin-fixed, paraffin-embedded glioblastoma tissues derived from patients treated in a prospective clinical phase II trial that tested up-front chemoradiotherapy with dose-intensified temozolomide (UKT-05). Our goal was to determine which of these three diagnostic methods provides the most accurate prediction of progression-free survival (PFS). The MGMT promoter methylation status was assessable by each method in almost all cases (n = 33/35 for MSP; n = 35/35 for PSQ; n = 34/35 for MS-MLPA). We were able to calculate significant cut-points for the continuous methylation signals at each CpG site analysed by PSQ (range, 11.5 to 44.9%) and at one CpG site assessed by MS-MLPA (3.6%) indicating that a dichotomisation of continuous methylation data as a prerequisite for comparative survival analyses is feasible. Our results show that, unlike MS-MLPA, MSP and PSQ provide a significant improvement of predicting PFS compared with established clinical prognostic factors alone (likelihood ratio tests: p<0.001). Conclusively, taking into consideration prognostic value, cost effectiveness and ease of use, we recommend pyrosequencing for analyses of MGMT promoter methylation in high-throughput settings and MSP for clinical routine diagnostics with low sample numbers.
authors.affiliation: Clinical Cooperation Unit Neuropathology, German Cancer Research Center, DKFZ, Heidelberg, Germany.
authors.forename: Arne
authors.initials: A
authors.lastname: Christians
authors.forename: Christian
authors.initials: C
authors.lastname: Hartmann
authors.forename: Axel
authors.initials: A
authors.lastname: Benner
authors.forename: Jochen
authors.initials: J
authors.lastname: Meyer
authors.forename: Andreas
authors.initials: A
authors.lastname: von Deimling
authors.forename: Michael
authors.initials: M
authors.lastname: Weller
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wick
authors.forename: Markus
authors.initials: M
authors.lastname: Weiler
mesh_terms: D019175:DNA Methylation / Q000235:genetics*; D015254:DNA Modification Methylases / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D045643:DNA Repair Enzymes / Q000235:genetics* / Q000378:metabolism; D003606:Dacarbazine / Q000031:analogs & derivatives; D018572:Disease-Free Survival; D005909:Glioblastoma / Q000175:diagnosis* / Q000188:drug therapy / Q000235:genetics; D006801:Humans; D053208:Kaplan-Meier Estimate; D016013:Likelihood Functions; D015336:Molecular Probe Techniques; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011446:Prospective Studies; D017422:Sequence Analysis, DNA / Q000379:methods*; D000077204:Temozolomide; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D025521:Tumor Suppressor Proteins; D003606:Dacarbazine; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes; D000077204:Temozolomide