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

// Véronique Quillien 1 , Audrey Lavenu 2, 3 , François Ducray 4, 5 , Marie-Odile Joly 4, 5 , Olivier Chinot 6 , Frédéric Fina 7 , Marc Sanson 8 , Catherine Carpentier 8 , Lucie Karayan-Tapon 9, 10 , Pierre Rivet 10 , Natacha Entz-Werle 11, 12 , Michèle Legrain 11 , Emmanuèle Lechapt Zalcman 13 , Guenaelle Levallet 13 , Fabienne Escande 14 , Carole Ramirez 14 , Dan Chiforeanu 15 , Elodie Vauleon 1 , Dominique Figarella-Branger 6, 16 1 Centre Eugène Marquis, F-35042 Rennes, France 2 Université Rennes 1, Faculté de Médecine, F-35043 Rennes, France 3 INSERM CIC 0203, Université de Rennes 1, F-35043 Rennes, France 4 Hospices Civils de Lyon, F- 69394, Lyon, Cedex, France 5 Université de Lyon1, F-69622 Villeurbanne, France 6 CHU Timone, F-13385 Marseille, France 7 Faculté de Médecine Secteur Nord, F-13916 Marseille, France 8 Sorbonne Universités UPMC Université Paris 06, INSERM CNRS, U1127, UMR 7225, ICM, F-75013 Paris, France 9 INSERM U1084, Université de Poitiers, F-86021 Poitiers, France 10 CHU de Poitiers, F-86021 Poitiers, France 11 CHRU Hautepierre, F67098 Strasbourg, France 12 EA 3430, Progression Tumorale et Microenvironnement, Approches Translationnelles et Épidémiologie, Université de Strasbourg, F-67000 Strasbourg, France 13 CHU Caen, Département de Pathologie, F-14000 Caen, France 14 CHRU de Lille, F-59037 Lille, France 15 CHU de Rennes, F-35000 Rennes, France 16 INSERM U911 CRO2, Université de la Méditerranée, F-13385 Marseille, France Correspondence to: Véronique Quillien, email: v.quillien@rennes.unicancer.fr Keywords: glioblastoma, prospective trial, MGMT, promoter methylation, pyrosequencing Received: May 18, 2016     Accepted: July 28, 2016     Published: August 17, 2016 ABSTRACT 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.