[Analysis of MGMT methylation with the therascreen(®) MGMT Pyro(®) Kit (Qiagen). A method verification].
Fiche publication
Journal
Annales de biologie clinique
Auteurs
Membres identifiés du Cancéropôle Est :
Pr PRETET Jean-Luc, Dr GUENAT David, Dr MAGNIN Sandrine
Tous les auteurs :
Luquain A, Magnin S, Guenat D, Prétet JL, Viennet G, Valmary-Degano S, Mougin C
Lien Pubmed
Résumé
Promoter methylation of the MGMT gene, encoding the enzyme O6-methylguanine-ubiquitous methyltransferase, is a theranostic good prognosis marker of glioblastomas treated with alkylating chemotherapy (temozolomide, Temodal(®)). Among the methylation analysis techniques, pyrosequencing is a reproducible and sensitive quantitative method. As part of the accreditation of the hospital platform of molecular genetics of cancer, Besançon, our objective was to verify the performance of the pyrosequencing commercial kit therascreen(®) MGMT Pyro(®) (Qiagen) in terms of repeatability, reproducibility, limit of blank (LOB), limit of detection (LOD), linearity and contamination by the guide SH GTA 04 delivered by the Cofrac. The repeatability tests show an average methylation of 3.22% [standard deviation (SD) = 0.41, coefficient of variation (CV) = 12.75%] for the unmethylated control and 70.16% (SD = 2.20, CV = 3.14%) for the methylated control. Reproducibility demontrates an average methylation of 1.39% (SD = 0.25, CV = 18.25%) for the unmethylated control and of 94.03% (SD = 2.56, CV = 2.73%) for the methylated control. The percentages of LOB and LOD are respectively 3.43% and 6.22% methylation. The regression coefficient of 0,983 confirms the linearity of the assay from 0% to 100% methylation. No contamination has been observed. Over 40% of glioblastomas studied in 2013 in our laboratory have shown a methylated MGMT gene. Our results confirms that the theraScreen(®) MGMT Pyro(®) kit (Qiagen) is performant in compliance with the quality requirements of the NF EN ISO 15189 for the routine analysis of methylation status of MGMT in glioblastomas.
Mots clés
Accreditation, Antineoplastic Agents, Alkylating, therapeutic use, Biomarkers, Tumor, analysis, Brain Neoplasms, diagnosis, DNA Methylation, DNA Modification Methylases, genetics, DNA Repair Enzymes, genetics, Dacarbazine, analogs & derivatives, Glioblastoma, diagnosis, Humans, Molecular Diagnostic Techniques, standards, Monitoring, Physiologic, methods, Prognosis, Promoter Regions, Genetic, Reagent Kits, Diagnostic, Reproducibility of Results, Sequence Analysis, DNA, methods, Treatment Outcome, Tumor Suppressor Proteins, genetics
Référence
Ann. Biol. Clin. (Paris). ;73(6):665-70