Dataset Information

Clinical Neuropathology practice guide 5-2015: MGMT methylation pyrosequencing in glioblastoma: unresolved issues and open questions.

ABSTRACT: O6-methylguanine-methyltransferase (MGMT) promoter methylation status has prognostic and, in the subpopulation of elderly patients, predictive value in newly diagnosed glioblastoma. Therefore, knowledge of the MGMT promoter methylation status is important for clinical decision-making. So far, MGMT testing has been limited by the lack of a robust test with sufficiently high analytical performance. Recently, one of several available pyrosequencing protocols has been shown to be an accurate and robust method for MGMT testing in an intra- and interlaboratory ring trial. However, some uncertainties remain with regard to methodological issues, cut-off definitions, and optimal use in the clinical setting. In this article, we highlight and discuss several of these open questions. The main unresolved issues are the definition of the most relevant CpG sites to analyze for clinical purposes and the determination of a cut-off value for dichotomization of quantitative MGMT pyrosequencing results into "MGMT methylated" and "MGMT unmethylated" patient subgroups as a basis for further treatment decisions.

SUBMITTER: Bienkowski M

PROVIDER: S-EPMC4542181 | biostudies-other | 2015 Sep-Oct

REPOSITORIES: biostudies-other

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Clinical Neuropathology practice guide 5-2015: MGMT methylation pyrosequencing in glioblastoma: unresolved issues and open questions.

Bienkowski Michal M Berghoff Anna S AS Marosi Christine C Wöhrer Adelheid A Heinzl Harald H Hainfellner Johannes A JA Preusser Matthias M

Clinical neuropathology 20150901 5

O6-methylguanine-methyltransferase (MGMT) promoter methylation status has prognostic and, in the subpopulation of elderly patients, predictive value in newly diagnosed glioblastoma. Therefore, knowledge of the MGMT promoter methylation status is important for clinical decision-making. So far, MGMT testing has been limited by the lack of a robust test with sufficiently high analytical performance. Recently, one of several available pyrosequencing protocols has been shown to be an accurate and rob ...[more]

PMID: 26295302

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Project description:The cyclooxygenase and peroxidase activities of prostaglandin H synthase (PGHS)-1 and -2 have complex kinetics, with the cyclooxygenase exhibiting feedback activation by product peroxide and irreversible self-inactivation, and the peroxidase undergoing an independent self-inactivation process. The mechanistic bases for these complex, non-linear steady-state kinetics have been gradually elucidated by a combination of structure/function, spectroscopic and transient kinetic analyses. It is now apparent that most aspects of PGHS-1 and -2 catalysis can be accounted for by a branched chain radical mechanism involving a classic heme-based peroxidase cycle and a radical-based cyclooxygenase cycle. The two cycles are linked by the Tyr385 radical, which originates from an oxidized peroxidase intermediate and begins the cyclooxygenase cycle by abstracting a hydrogen atom from the fatty acid substrate. Peroxidase cycle intermediates have been well characterized, and peroxidase self-inactivation has been kinetically linked to a damaging side reaction involving the oxyferryl heme oxidant in an intermediate that also contains the Tyr385 radical. The cyclooxygenase cycle intermediates are poorly characterized, with the exception of the Tyr385 radical and the initial arachidonate radical, which has a pentadiene structure involving C11-C15 of the fatty acid. Oxygen isotope effect studies suggest that formation of the arachidonate radical is reversible, a conclusion consistent with electron paramagnetic resonance spectroscopic observations, radical trapping by NO, and thermodynamic calculations, although moderate isotope selectivity was found for the H-abstraction step as well. Reaction with peroxide also produces an alternate radical at Tyr504 that is linked to cyclooxygenase activation efficiency and may serve as a reservoir of oxidizing equivalent. The interconversions among radicals on Tyr385, on Tyr504, and on arachidonate, and their relationships to regulation and inactivation of the cyclooxygenase, are still under active investigation for both PGHS isozymes.

Dataset Information

Clinical Neuropathology practice guide 5-2015: MGMT methylation pyrosequencing in glioblastoma: unresolved issues and open questions.

Publications

Clinical Neuropathology practice guide 5-2015: MGMT methylation pyrosequencing in glioblastoma: unresolved issues and open questions.

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