MGMT promoter methylation in malignant gliomas: ready for personalized medicine?

TLDR: The prognostic and predictive value of MGMT silencing is critically discussed, reviewing trials in which patients with different types of glioma were treated with various chemotherapy schedules, either upfront or at recurrence.

Abstract: The DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) antagonizes the genotoxic effects of alkylating agents MGMT promoter methylation is the key mechanism of MGMT gene silencing and predicts a favorable outcome in patients with glioblastoma who are exposed to alkylating agent chemotherapy This biomarker is on the verge of entering clinical decision-making and is currently used to stratify or even select glioblastoma patients for clinical trials In other subtypes of glioma, such as anaplastic gliomas, the relevance of MGMT promoter methylation might extend beyond the prediction of chemosensitivity, and could reflect a distinct molecular profile Here, we review the most commonly used assays for evaluation of MGMT status, outline the prerequisites for standardized tests, and evaluate reasons for difficulties in reproducibility We critically discuss the prognostic and predictive value of MGMT silencing, reviewing trials in which patients with different types of glioma were treated with various chemotherapy schedules, either up-front or at recurrence Standardization of MGMT testing requires comparison of different technologies across laboratories and prospectively validated cut-off values for prognostic or predictive effects Moreover, future clinical trials will need to determine, for each subtype of glioma, the degree to which MGMT promoter methylation is predictive or prognostic, and whether testing should become routine clinical practice

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University of Zurich
Zurich Open Repository and Archive
Winterthurerstr. 190
CH-8057 Zurich
http://www.zora.uzh.ch
Year: 2010
MGMT promoter methylation in malignant gliomas: ready for
personalized medicine?
Weller, M; Stupp, R; Reifenberger, G; Brandes, A A; van den Bent, M J; Wick, W;
Hegi, M E
Weller, M; Stupp, R; Reifenberger, G; Brandes, A A; van den Bent, M J; Wick, W; Hegi, M E (2010). MGMT
promoter methylation in malignant gliomas: ready for personalized medicine? Nature Reviews Neurology,
6(1):39-51.
Postprint available at:
http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.
http://www.zora.uzh.ch
Originally published at:
Nature Reviews Neurology 2010, 6(1):39-51.
Weller, M; Stupp, R; Reifenberger, G; Brandes, A A; van den Bent, M J; Wick, W; Hegi, M E (2010). MGMT
promoter methylation in malignant gliomas: ready for personalized medicine? Nature Reviews Neurology,
6(1):39-51.
Postprint available at:
http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.
http://www.zora.uzh.ch
Originally published at:
Nature Reviews Neurology 2010, 6(1):39-51.

MGMT promoter methylation in malignant gliomas: ready for personalized
medicine?
Michael Weller, Roger Stupp, Guido Reifenberger, Alba A. Brandes, Martin J. van den
Bent, Wolfgang Wick and Monika E. Hegi
ABSTRACT
The DNA repair enzyme O
6
-methylguanine-DNA methyltransferase (MGMT)
antagonizes the genotoxic effects of alkylating agents. MGMT promoter methylation is
the key mechanism of MGMT gene silencing and predicts a favorable outcome in
patients with glioblastoma who are exposed to alkylating agent chemotherapy. This
biomarker is on the verge of entering clinical decision-making and is currently used to
stratify or even select glioblastoma patients for clinical trials. In other subtypes of glioma,
such as anaplastic gliomas, the relevance of MGMT promoter methylation might extend
beyond the prediction of chemosensitivity, and could reflect a distinct molecular profile.
Here, we review the most commonly used assays for evaluation of MGMT status, outline
the prerequisites for standardized tests, and evaluate reasons for difficulties in
reproducibility. We critically discuss the prognostic and predictive value of MGMT
silencing, reviewing trials in which patients with different types of glioma were treated
with various chemotherapy schedules, either upfront or at recurrence. Standardization of
MGMT testing requires comparison of different technologies across laboratories, and
prospectively validated cut-off values for prognostic or predictive effects. Moreover,
future clinical trials will need to determine, for each subtype of glioma, the degree to
which MGMT promoter methylation is predictive or prognostic, and whether testing
should become routine clinical practice.

2
Weller, M. et al. Nat. Rev. Neurol. advance online publication XX Month 2009;
doi:10.1038/
Department of Neurology, University Hospital Zurich, Frauenklinikstrasse 26, CH-8091
Zurich, Switzerland (M. Weller).
Department of Neurosurgery, Centre Hospitalier
Universitaire Vaudois and University of Lausanne, Rue du Bugnon 46, CH-1011
Lausanne, Switzerland (R. Stupp, M . E. Hegi). Department of Neuropathology,
Heinrich-Heine-University, Moorenstrasse 5, D-40225 Düsseldorf, Germany (G.
Reifenberger).
Department of Medical Oncology, Bellaria Hospital, Via Altura 3, 40139
Bologna, Italy (A. A. Brandes).
Neuro-Oncology Unit, Daniel de Hoed Cancer
Centre/Erasmus, University Hospital Rotterdam, 3008AE Rotterdam, The Netherlands
(M. J. van den Bent).Department of Neurooncology, University of Heidelberg, Im
Neuenheimer Feld 400, D-69120 Heidelberg, Germany (W. Wick).
Correspondence to: M. Weller, michael.weller@usz.ch
Competing interests
M. Weller, R. Stupp and M. E. Hegi declare associations with the following companies:
Merck Serono, OncoMethylome Sciences, Schering-Plough. G. Reifenberger declares
associations with the following companies: Essex Pharma, OncoMethylome Sciences,
Miltenyi Biotec. A. A. Brandes declares associations with the following companies:
OncoMethylome Sciences, Schering-Plough. M. van den Bent declares associations
with the following companies: Merck AG, Oncomethylome Sciences, Schering-Plough.

3
W. Wick declares associations with the following companies: Merck Serono, Schering-
Plough. See the article online for full details of the relationships.
Key points
 MGMT (O
6
-methylguanine-DNA methyltransferase) promoter methylation has
become the most powerful molecular prognosticator in malignant gliomas
 MGMT promoter methylation is predictive for response to alkylating agent
chemotherapy in glioblastoma
 Methylation-specific PCR is the only validated technique to derive prognostic
information from determination of the MGMT status
 The MGMT status has become a parameter for stratification of patients with
glioma within clinical trials

4
INTRODUCTION
O
6
-methylguanine-DNA methyltransferase (MGMT) is a ubiquitous DNA repair enzyme
that has been highly conserved throughout evolution. MGMT is associated with
resistance to alkylating agent cancer therapy, and modulation of this enzyme as a
treatment target has been under investigation for over 2 decades.
1,2
MGMT rapidly
reverses alkylation, including methylation, at the O
6
position of guanine by transferring
the alkyl group to the active site of the enzyme.
3
Although O
6
-alkylguanine is not the
main lesion induced by alkylating agents, it seems to be the most cytotoxic one. Lack of
MGMT in the cell allows accumulation of O
6
-alkylguanine in the DNA, which, subsequent
to incorrect pairing with thymidine, triggers mismatch repair, thereby inducing DNA
damage signaling and, eventually, cell death.
4,5
In accordance with this postulated
mechanism, mismatch repair-deficient cells are highly resistant to alkylating agents,
even in the absence of MGMT.
In this article, we critically review the prognostic and predictive value of MGMT
silencing in gliomas, drawing on the results of trials in which various chemotherapy
schedules were used to treat patients with these tumors. We discuss the assays that are
most commonly used to evaluate MGMT status, outline the prerequisites for
standardized tests, and consider possible reasons for difficulties in reproducibility.
THE MGMT GENE AND ITS PROMOTER
The MGMT gene is located on chromosome 10q26. Its promoter lacks the constitutive
regulatory elements known as the TATA box and the CAT box, similar to many
housekeeping genes, and contains a CpG island. CpG islands are genomic regions,
typically of 300–3000 bp, that contain a high frequency of CG dinucleotides (CpG sites),

Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "Mgmt promoter methylation in malignant gliomas: ready for personalized medicine?" ?

Here, the authors review the most commonly used assays for evaluation of MGMT status, outline the prerequisites for standardized tests, and evaluate reasons for difficulties in reproducibility. The authors critically discuss the prognostic and predictive value of MGMT silencing, reviewing trials in which patients with different types of glioma were treated with various chemotherapy schedules, either upfront or at recurrence. 

Q2. What are the commonly used technologies to analyze bisulfite-converted DNA?

Recently developed technologies that analyze bisulfite-converted DNA include methylation-sensitive highresolution melting (MS-HRM; a PCR-based method that differentiates the melting behavior of the amplicons derived from methylated and unmethylated sequences),34 bead array-based technologies,35 mass spectroscopy,36 and denaturing highperformance liquid chromatography. 

Q3. What is the reason for the small improvement in outcome in MGMT?

The small improvement in outcome even in the patients with an unmethylated MGMT promoter could be attributable to the above-mentioned gray zone separating methylated and unmethylated tumors, and the consequent somewhat arbitrary separation into two groups. 

Q4. What is the common technique used for methylation-specific assays?

Thistechnique uses methylation-specific primers, each of which is designed to bind only to completely methylated or unmethylated sequences. 

Q5. What protocol should be used for MGMT testing outside of the academic context?

Methylation testing performed outside the academic trial context should also follow a specified protocol that lends itself to independent reproduction. 

Q6. What is the role of temozolomide in MGMT?

Since temozolomide is itself a substrate for MGMT, alternative, more-protracteddosing regimes of temozolomide have been explored in first-line and recurrent22glioblastoma settings. 

Q7. What is the reason why a methylated MGMT promoter should not be used?

Since MGMT promoter methylation is prognostic and not predictive for chemotherapy response in anaplastic gliomas, a methylated MGMT promoter should not be used to justify the upfront treatment of these tumors with temozolomide-based radiochemotherapy in the absence of appropriate data from studies such as CATNON (http://clinicaltrials.gov, NCT00626990). 

Q8. What is the principle for the discrimination of unmethylated from methylated sequences?

The principle for the discrimination of unmethylated from methylated sequences that is used by most methylation-specific assays is based on a bisulfite treatment step that converts unmethylated cytidine—but not 5-methylcytidine—in the DNA to uracil (Figure 4). 

Q9. What is the common method for detecting methylated and unmethylated sequences?

Subsequent detection and quantification of the methylated and unmethylated sequences can be performed by various technologies to create semiquantitative or quantitative assays (Table 1). 

Q10. When did the first observations on a potential predictive value of MGMT protein be made?

The first striking observations on a potential predictive value of MGMT protein levels, as determined by immunofluorescence microscopy, in patients with malignant glioma were made more than 10 years ago. 

Q11. What is the role of MGMT in the progression-free survival of gliom?

low levels of MGMT protein, as detected by immunohistochemistry, predicted prolonged progression-free survival (PFS) in patients with glioma treated upfront with temozolomide,8 or prolonged overall survival in patients with newly diagnosed, inoperable glioblastoma treated with neoadjuvant temozolomide.