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Oncotarget. 2016 Sep 20;7(38):61916-61929. doi: 10.18632/oncotarget.11322.

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

Author information

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.

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.

KEYWORDS:

MGMT; glioblastoma; promoter methylation; prospective trial; pyrosequencing

PMID:
27542245
PMCID:
PMC5308700
DOI:
10.18632/oncotarget.11322
[Indexed for MEDLINE]
Free PMC Article

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