title: Validation of the high-performance of pyrosequencing for clinical MGMT testing on a cohort of glioblastoma patients from a prospective dedicated multicentric trial.
abstract: The goal of this prospective multicentric trial was to validate a technique that allowed for MGMT promoter methylation analysis in routine clinical practice.
  
  
  
  The MGMT status of 139 glioblastoma patients, whom had received standard first line treatment, was determined using pyrosequencing (PSQ) and a semi-quantitative Methylation-specific PCR (sqMS-PCR) method, using both frozen and formalin-fixed paraffin-embedded FFPE samples. Eight participating centers locally performed the analysis, including external quality controls.
  
  
  
  There was a strong correlation between results from FFPE and frozen samples. With cut-offs of 12% and 13%, 98% and 91% of samples were identically classified with PSQ and sqMS-PCR respectively. In 12% of cases frozen samples were excluded because they had a low percentage of tumor cells. In 5-6% of cases the analysis was not feasible on FFPE samples. The optimized risk cut-offs were higher in both techniques when using FFPE samples, in comparison to frozen samples. For sqMS-PCR, we validated a cut-off between 13-15% to dichotomize patients. For PSQ, patients with a low level of methylation (<= 8%) had a median progression-free survival under 9 months, as compared with more than 15.5 months for those with a level above 12%. For intermediate values (9-12%), more discordant results between FFPE and frozen samples were observed and there was not a clear benefit of temozolomide treatment, which indicated a "grey zone".
  
  
  
  MGMT status can reliably be investigated in local laboratories. PSQ is the ideal choice as proven by strong interlaboratory reproducibility, along with threshold agreements across independent studies.
authors.affiliation: Centre Eugène Marquis, F-35042 Rennes, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Quillien
authors.affiliation: Université Rennes 1, Faculté de Médecine, F-35043 Rennes, France.|INSERM CIC 0203, Université de Rennes 1, F-35043 Rennes, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Lavenu
authors.affiliation: Hospices Civils de Lyon, F- 69394, Lyon, Cedex, France.|Université de Lyon1, F-69622 Villeurbanne, France.
authors.forename: François
authors.initials: F
authors.lastname: Ducray
authors.affiliation: Hospices Civils de Lyon, F- 69394, Lyon, Cedex, France.|Université de Lyon1, F-69622 Villeurbanne, France.
authors.forename: Marie-Odile
authors.initials: MO
authors.lastname: Joly
authors.affiliation: CHU Timone, F-13385 Marseille, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Chinot
authors.affiliation: Faculté de Médecine Secteur Nord, F-13916 Marseille, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Fina
authors.affiliation: Sorbonne Universités UPMC Université Paris 06, INSERM CNRS, U1127, UMR 7225, ICM, F-75013 Paris, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Sanson
authors.affiliation: Sorbonne Universités UPMC Université Paris 06, INSERM CNRS, U1127, UMR 7225, ICM, F-75013 Paris, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Carpentier
authors.affiliation: INSERM U1084, Université de Poitiers, F-86021 Poitiers, France.|CHU de Poitiers, F-86021 Poitiers, France.
authors.forename: Lucie
authors.initials: L
authors.lastname: Karayan-Tapon
authors.affiliation: CHU de Poitiers, F-86021 Poitiers, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Rivet
authors.affiliation: CHRU Hautepierre, F67098 Strasbourg, France.|EA 3430, Progression Tumorale et Microenvironnement, Approches Translationnelles et Épidémiologie, Université de Strasbourg, F-67000 Strasbourg, France.
authors.forename: Natacha
authors.initials: N
authors.lastname: Entz-Werle
authors.affiliation: CHRU Hautepierre, F67098 Strasbourg, France.
authors.forename: Michèle
authors.initials: M
authors.lastname: Legrain
authors.affiliation: CHU Caen, Département de Pathologie, F-14000 Caen, France.
authors.forename: Emmanuèle Lechapt
authors.initials: EL
authors.lastname: Zalcman
authors.affiliation: CHU Caen, Département de Pathologie, F-14000 Caen, France.
authors.forename: Guenaelle
authors.initials: G
authors.lastname: Levallet
authors.affiliation: CHRU de Lille, F-59037 Lille, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Escande
authors.affiliation: CHRU de Lille, F-59037 Lille, France.
authors.forename: Carole
authors.initials: C
authors.lastname: Ramirez
authors.affiliation: CHU de Rennes, F-35000 Rennes, France.
authors.forename: Dan
authors.initials: D
authors.lastname: Chiforeanu
authors.affiliation: Centre Eugène Marquis, F-35042 Rennes, France.
authors.forename: Elodie
authors.initials: E
authors.lastname: Vauleon
authors.affiliation: CHU Timone, F-13385 Marseille, France.|INSERM U911 CRO2, Université de la Méditerranée, F-13385 Marseille, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Figarella-Branger
mesh_terms: D000328:Adult; D000368:Aged; D001932:Brain Neoplasms / Q000235:genetics*; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics*; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000627:therapeutic use; D018572:Disease-Free Survival; D005260:Female; D005909:Glioblastoma / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D011446:Prospective Studies; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D000077204:Temozolomide; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D055815:Young Adult
chemical_list: D025521:Tumor Suppressor Proteins; D003606:Dacarbazine; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes; D000077204:Temozolomide
keywords: MGMT; glioblastoma; promoter methylation; prospective trial; pyrosequencing