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

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Oncotarget. 2016; 7:61916-61929. https://doi.org/10.18632/oncotarget.11322

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Abstract

Véronique Quillien1, Audrey Lavenu2,3, François Ducray4,5, Marie-Odile Joly4,5, Olivier Chinot6, Frédéric Fina7, Marc Sanson8, Catherine Carpentier8, Lucie Karayan-Tapon9,10, Pierre Rivet10, Natacha Entz-Werle11,12, Michèle Legrain11, Emmanuèle Lechapt Zalcman13, Guenaelle Levallet13, Fabienne Escande14, Carole Ramirez14, Dan Chiforeanu15, Elodie Vauleon1, Dominique Figarella-Branger6,16

1Centre Eugène Marquis, F-35042 Rennes, France

2Université Rennes 1, Faculté de Médecine, F-35043 Rennes, France

3INSERM CIC 0203, Université de Rennes 1, F-35043 Rennes, France

4Hospices Civils de Lyon, F- 69394, Lyon, Cedex, France

5Université de Lyon1, F-69622 Villeurbanne, France

6CHU Timone, F-13385 Marseille, France

7Faculté de Médecine Secteur Nord, F-13916 Marseille, France

8Sorbonne Universités UPMC Université Paris 06, INSERM CNRS, U1127, UMR 7225, ICM, F-75013 Paris, France

9INSERM U1084, Université de Poitiers, F-86021 Poitiers, France

10CHU de Poitiers, F-86021 Poitiers, France

11CHRU Hautepierre, F67098 Strasbourg, France

12EA 3430, Progression Tumorale et Microenvironnement, Approches Translationnelles et Épidémiologie, Université de Strasbourg, F-67000 Strasbourg, France

13CHU Caen, Département de Pathologie, F-14000 Caen, France

14CHRU de Lille, F-59037 Lille, France

15CHU de Rennes, F-35000 Rennes, France

16INSERM U911 CRO2, Université de la Méditerranée, F-13385 Marseille, France

Correspondence to:

Véronique Quillien, email: [email protected]

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.


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