title: [Analysis of MGMT methylation with the therascreen(®) MGMT Pyro(®) Kit (Qiagen). A method verification].
abstract: 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.
authors.affiliation: Service d'anatomie et cytologie pathologiques et biologie cellulaire et moléculaire, CHRU de Besançon et Université de Franche-Comté, Besançon, France.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Luquain
authors.affiliation: Service d'anatomie et cytologie pathologiques et biologie cellulaire et moléculaire, CHRU de Besançon et Université de Franche-Comté, Besançon, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Magnin
authors.affiliation: Service d'anatomie et cytologie pathologiques et biologie cellulaire et moléculaire, CHRU de Besançon et Université de Franche-Comté, Besançon, France, UMR 1098, Inserm, Besançon.
authors.forename: David
authors.initials: D
authors.lastname: Guenat
authors.affiliation: Service d'anatomie et cytologie pathologiques et biologie cellulaire et moléculaire, CHRU de Besançon et Université de Franche-Comté, Besançon, France, EA 3181, FED 4234, LabEx LipSTIC ANR-11-LABX-0021, Besançon.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Prétet
authors.affiliation: Service d'anatomie et cytologie pathologiques et biologie cellulaire et moléculaire, CHRU de Besançon et Université de Franche-Comté, Besançon, France.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Viennet
authors.affiliation: Service d'anatomie et cytologie pathologiques et biologie cellulaire et moléculaire, CHRU de Besançon et Université de Franche-Comté, Besançon, France, EA 3181, FED 4234, LabEx LipSTIC ANR-11-LABX-0021, Besançon.
authors.forename: Séverine
authors.initials: S
authors.lastname: Valmary-Degano
authors.affiliation: Service d'anatomie et cytologie pathologiques et biologie cellulaire et moléculaire, CHRU de Besançon et Université de Franche-Comté, Besançon, France, EA 3181, FED 4234, LabEx LipSTIC ANR-11-LABX-0021, Besançon.
authors.forename: Christiane
authors.initials: C
authors.lastname: Mougin
mesh_terms: D000068:Accreditation; D018906:Antineoplastic Agents, Alkylating / Q000627:therapeutic use; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics; D001932:Brain Neoplasms / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics* / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000235:genetics* / Q000378:metabolism; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000627:therapeutic use; D005909:Glioblastoma / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000592:standards; D008991:Monitoring, Physiologic / Q000379:methods; D011379:Prognosis; D011401:Promoter Regions, Genetic; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods / Q000592:standards; D000077204:Temozolomide; D016896:Treatment Outcome; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018906:Antineoplastic Agents, Alkylating; D014408:Biomarkers, Tumor; D011933:Reagent Kits, Diagnostic; D025521:Tumor Suppressor Proteins; D003606:Dacarbazine; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes; D000077204:Temozolomide
keywords: ISO 15189; MGMT; methylation; on-site verification; pyrosequencing; validation method