Validation of the high-performance of pyrosequencing for clinical MGMT testing on a cohort of glioblastoma patients from a prospective dedicated multicentric trial


  • Quillien Véronique
  • Lavenu Audrey
  • Ducray François
  • Joly Marie-Odile
  • Chinot Olivier
  • Fina Frédéric
  • Sanson Marc
  • Carpentier Catherine
  • Karayan-Tapon Lucie
  • Rivet Pierre
  • Entz-Werle Natacha
  • Legrain Michèle
  • Lechapt Zalcman Emmanuèle
  • Levallet Guenaelle
  • Escande Fabienne
  • Ramirez Carole
  • Chiforeanu Dan
  • Vauleon Elodie
  • Figarella-Branger Dominique


  • Promoter methylation
  • Glioblastoma
  • MGMT
  • Pyrosequencing
  • Prospective trial

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Background: The goal of this prospective multicentric trial was to validate a technique that allowed for MGMT promoter methylation analysis in routine clinical practice.Methods: 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.Results: 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”.Conclusions: 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.

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