Neuro-Oncology 1457

23(9), 1457–1469, 2021 | doi:10.1093/neuonc/noab105 | Advance Access date 30 April 2021

MGMT promoter methylation testing to predict overall

survival in people with glioblastoma treated with
temozolomide: a comprehensive meta-analysis based
on a Cochrane Systematic Review
  

Downloaded from https://academic.oup.com/neuro-oncology/article/23/9/1457/6263562 by guest on 18 April 2023

Sebastian Brandner , Alexandra McAleenan , Claire Kelly, Francesca Spiga , Hung-Yuan Cheng ,
Sarah Dawson, Lena Schmidt , Claire L. Faulkner, Christopher Wragg , Sarah Jefferies ,
Julian P. T. Higgins , and Kathreena M. Kurian

Division of Neuropathology and Department of Neurodegenerative Disease, UCL Queen Square Institute of
Neurology, University College London, London, UK (S.B.); Population Health Sciences, Bristol Medical School,
University of Bristol, Bristol, UK (A.M., C.K., F.S., H.Y.C., S.D., L.S., J.P.T.H.); Bristol Genetics Laboratory, Pathology
Sciences, Southmead Hospital, Bristol, UK (C.L.F., C.W.); Department of Oncology, Addenbrooke’s Hospital,
Cambridge, UK (S.F.); Bristol Medical School, Brain Tumour Research Centre, Population Health Sciences, University
of Bristol, Bristol, UK (K.M.K.)

“This article is based on a Cochrane Review published in the Cochrane Database of Systematic Reviews (CDSR): Cochrane Database
Syst Rev. 2021;3:CD013316. doi: 10.1002/14651858.CD013316.pub2.”

Corresponding Authors: Dr Kathreena M. Kurian, BSc, MD, MBBS, FRCPath(Neuro), Honorary Consultant Neuropathologist/
Associate Professor in Brain Tumour Research, Population Health Sciences, Bristol Medical School, Southmead Hospital, University
of Bristol/North Bristol Trust, Southmead Road, Bristol BS10 5NB, UK (kathreena.kurian@bristol.ac.uk); Sebastian Brandner, MD,
FRCPath, Professor of Neuropathology, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology,
Honorary Consultant Neuropathologist, The National Hospital for Neurology and Neurosurgery, University College London Hospitals
NHS Foundation Trust, Room 103 Level 1, Mailbox 126, Queen Square House, Queen Square, London WC1N 3BG, UK (s.brandner@
ucl.ac.uk).

Abstract
Background.  The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) causes resistance of
tumor cells to alkylating agents. It is a predictive biomarker in high-grade gliomas treated with temozolomide,
however, there is no consensus on which test method, methylation sites, and cutoff values to use.
Methods.  We performed a Cochrane Review to examine studies using different techniques to measure MGMT
and predict survival in glioblastoma patients treated with temozolomide. Eligible longitudinal studies included (i)
adults with glioblastoma treated with temozolomide with or without radiotherapy, or surgery; (ii) where MGMT
status was determined in tumor tissue, and assessed by 1 or more technique; and (iii) where overall survival was
an outcome parameter, with sufficient information to estimate hazard ratios (HRs). Two or more methods were
compared in 32 independent cohorts with 3474 patients.
Results.  Methylation-specific PCR (MSP) and pyrosequencing (PSQ) techniques were more prognostic than
immunohistochemistry for MGMT protein, and PSQ is a slightly better predictor than MSP.
Conclusions.  We cannot draw strong conclusions about use of frozen tissue vs formalin-fixed paraffin-embedded
in MSP and PSQ. Also, our meta-analysis does not provide strong evidence about the best CpG sites or threshold.
MSP has been studied mainly for CpG sites 76-80 and 84-87 and PSQ at CpG sites ranging from 72 to 95. A cutoff
threshold of 9% for CpG sites 74-78 performed better than higher thresholds of 28% or 29% in 2 of the 3 good-
quality studies. About 190 studies were identified presenting HRs from survival analysis in patients in which MGMT
methylation was measured by 1 technique only.

© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/
licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
For commercial re-use, please contact journals.permissions@oup.com
1458 Brandner et al. Meta-analysis of the predictive value of MGMT promoter methylation
Key Points
• Largest meta-analysis of predictive value of MGMT test methods, cutoff
methylated/unmethylated status, and CpG sites.
• Comparison of studies using 2 or more MGMT test methods.
• Methylation-specific PCR and pyrosequencing techniques best prognosticators.
Importance of the Study
It is important to reach a consensus of the best
method(s) for assessing MGMT methylation status,
based on the prognostic value of each method in
predicting overall survival in people with glioblas-
toma treated with temozolomide. Currently, there is no
consensus which CpG sites in the MGMT promoter
region to be analyzed and which are the most rele-
vant cutoffs to determine methylated vs unmethylated
status for quantitative tests. Previous systematic re-
views have assessed the prognostic value of MGMT
The IDH (isocitrate dehydrogenase) wild-type glioblastoma
(glioblastoma multiforme [GBM]) is the most common pri-
mary brain tumor in adults, with an annual incidence of
approximately 3/100 000 population. The standard therapy
is surgical resection followed by radiotherapy and adju-
vant treatment with temozolomide, an alkylating agent.
The median overall survival is 9.9  months for people
treated with surgery plus radiotherapy and 15  months for
people treated with surgery, radiotherapy, and chemo-
therapy.1 For people with IDH-mutant glioblastomas, me-
dian overall survival is 24  months for people treated with
surgery and radiotherapy, and 31 months for people treated
with surgery, radiotherapy, and chemotherapy.1 The cyto-
toxic effects of temozolomide are exerted by induction of
O6-methylguanine and are counteracted by the repair en-
zyme O6-methylguanine-DNA methyltransferase (MGMT).2
Expression of MGMT is highly regulated by epigenetic silen-
cing of the MGMT gene promoter and thus the MGMT pro-
moter methylation status is a widely used predictive marker
for high-grade gliomas undergoing therapy with alkylating
agents. However, MGMT methylation status does not al-
ways reflect gene expression, so the exact mechanism by
which MGMT promoter methylation improves response to
alkylating therapy is still unknown.
MGMT promoter methylation status testing is essen-
tial to inform treatment decisions in certain patients
with GBM. For example, treating elderly patients with
an unmethylated MGMT promoter with temozolomide
has been shown to be detrimental when single-agent
temozolomide chemotherapy was compared to radio-
therapy.3,4 On the basis of these findings, professional
bodies, such as the European Association for Neuro-
Oncology (EANO), recommend evaluation of MGMT
promoter methylation status in elderly people,5 and The
Downloaded from https://academic.oup.com/neuro-oncology/article/23/9/1457/6263562 by guest on 18 April 2023
promoter status assessed by a specific technique, for
example, by pyrosequencing or methylation-specific
PCR. However, no review has quantitatively determined
which method correlates best with prognosis (although
a previous study provided a narrative overview). In
our Cochrane Review, we address a research priority
question identified by the James Lind Alliance Neuro-
Oncology Priority Setting Partnership—an organization
joining academics, patients, carers, and clinicians to
set research priorities in different fields.
National Institute for Health and Care Excellence (NICE) re-
commends that all high-grade gliomas are tested.6 Most
non-elderly (aged under 65  years) people are currently
treated with temozolomide chemotherapy regardless of
MGMT promoter status, due to the lack of alternative treat-
ments.7 However, MGMT promoter status is still a useful
prognostic marker that may impact clinical management
and may also be used for recruitment into clinical trials for
novel therapies.
A number of methods have been established to as-
sess MGMT promoter methylation status: methylation-
specific PCR (MSP), quantitative (real-time) MSP, such
as MethyLight MSP, pyrosequencing (PSQ), bead array,
methylation-specific multiplex ligation-dependent probe
amplification (MS-MLPA)-PCR with high-resolution
melting (HRM), co-amplification at lower denaturation
temperature (COLD)-PCR, and digestion-based assays.
Immunohistochemical detection of the MGMT protein or
enzymatic activity has also been used as a proxy for meth-
ylation status. However, internationally accepted con-
sensus about the most appropriate diagnostic method
for MGMT promoter status is lacking.8 MSP was used to
assess MGMT promoter status in the landmark study by
Hegi et al.9 The choice of technique to assess MGMT pro-
moter status in practice also depends on the amount and
quality of the DNA sample(s) (eg, formalin-fixed paraffin-
embedded (FFPE) vs frozen tissue-derived DNA), the ro-
bustness and simplicity of the method, the availability of
equipment and reagents necessary for each of the tech-
niques, cost, and experience. In the last United Kingdom
National Quality Assessment (UK NEQAS) External Quality
Assessment report, of 18 UK laboratories, 10 used PSQ,
5 MSP, 2 HRM, and 1 MS-MLPA. MGMT promoter meth-
ylation can also be determined with Illumina bead chip
 Brandner et al. Meta-analysis of the predictive value of MGMT promoter methylation 1459

arrays, an increasingly popular method for brain tumor
classification based on the epigenetic profile.10,11 All tech-
niques can only interrogate methylation status in specific
regions within the MGMT promoter, and the effect of meth-
ylation status at different sites on prognosis is not well un-
derstood. In addition, some of the techniques quantify the
amount of methylation present, and there is no consensus
regarding the cutoff for categorizing methylation status.
We undertook a Cochrane Review12 to assess which way
of measuring methylation of the MGMT promoter best pre-
dicts survival when people with glioblastoma are treated
with temozolomide. The present article provides a sum-
Oncology
Neuro-
MS-MLPA; (vi) PCR with HRM; (vii) COLD-PCR; and (viii)
digestion-based assays. We also included studies as-
sessing (ix) MGMT expression (eg, immunohistochemistry
[IHC] for protein expression, (x) mRNA levels, or (xi) MGMT
enzymatic activity. Studies not reporting the test methods
were excluded. Studies had to report a hazard ratio (HR),
or data sufficient to allow a HR to be calculated. All tech-
niques are listed in Table 1.
Search Methodology

Downloaded from https://academic.oup.com/neuro-oncology/article/23/9/1457/6263562 by guest on 18 April 2023

mary of the key findings from the Cochrane Review.
Methods
Study Eligibility
Longitudinal studies of (i) adults (18 years and older) with
(ii) first occurrence or recurrent glioblastoma, (iii) treated
with temozolomide, and (iv) optionally concomitant and
adjuvant therapies in addition to temozolomide, such as
surgery or radiotherapy or both) (v) for whom the MGMT
status was assessed by 1 or more techniques on tumor
tissue, (vi) taken prior to treatment, but (vii) not in other
types of samples such as blood samples, or by neuro-
imaging, were eligible for inclusion. Forms of glioma other
than glioblastoma could be represented only if they consti-
tuted less than 10% of the total cases.
Eligible studies had to assess MGMT promoter meth-
ylation status in tumor tissue by 1 or more techniques.
Eligible techniques included, but were not restricted to, (i)
MSP; (ii) quantitative MSP (real-time PCR or MethyLight
methylation-specific quantitative PCR); (iii) methylation-
specific sequencing, including PSQ; (iv) bead array; (v)
Electronic searches were performed on the following data-
bases up to December 2018: Ovid MEDLINE, PubMed (NOT
MEDLINE), Ovid Embase, BIOSIS, and Web of Science
Conference Proceedings Citation Index (CPCI-S). No re-
strictions were applied to language or date of publication.
Other resources for searches were meeting abstracts from
the Society of Neuro-Oncology (SNO), EANO, and the
Japan Society for Neuro-Oncology (JSNO), retrieved via
the CPCI-S. We examined the reference lists of included
studies and of systematic reviews that have assessed the
prognostic value of MGMT promoter status overall43 or as
assessed by a specific technique; for example, by PSQ44 or
MSP.45
Study Selection and Data Extraction
We used EPPI-Reviewer 4 (https://eppi.ioe.ac.uk) for pro-
cesses of screening and selection of studies and for part of
the data extraction the review.46 Data were extracted and
further analyzed in Microsoft Excel. Two review authors
(“reviewers”) independently screened titles and abstracts
of all identified search results and determined whether
full texts should be retrieved. Then, 2 reviewers inde-
pendently assessed the full-text articles. Disagreements

  
Table 1  Summary of the Characteristics of the Included Studies Comparing 2 and More Techniques

Technique Abbreviation No. of Studies References

Pyrosequencing PSQ 20 13–27

Methylation-specific PCR MSP 17 10,13,16–19,21–37

Immunohistochemistry IHC 9 13,18–20,22,26,31,33,36,38,39

Quantitative MSP qMSP 8 13,16,19,20,24,29,37,40

PCR with high-resolution melting HRM-PCR 3 13,16,35

Bead array 2 10,41

PCR targeting mRNA PCR-mRNA 2 20,30,38

Methylation-specific multiplex ligation-dependent probe amplifica- MS-MLPA 1 34

tion
Methylation-specific restriction enzyme quantitative PCR MS-RE-qPCR 1 42

Methyl-beaming 1 42

Quantitative fluorescence immunohistochemistry QF-IHC (AQUA) 1 29

Double immunofluorescence 1 NS cohort15, RSD cohort15

qMSP combined with PSQ 1 22

qMSP combined with sequencing 1 27

Abbreviations: NS, Nordic Study; RSD, Region of Southern Denmark.

  
1460 Brandner et al. Meta-analysis of the predictive value of MGMT promoter methylation

were resolved either by consensus or by consulting a
third reviewer. A Preferred Reporting Items for Systematic
Reviews and Meta-Analyses (PRISMA) flow diagram was
established (Figure 1) to describe the flow of information
through the different phases of the review.
Full data extraction, risk-of-bias assessment, and syn-
thesis were performed on studies that evaluated MGMT
promoter methylation status using 2 or more methods,
enabling comparisons of methods to be made using
the same samples of patients. Two reviewers independ-
ently performed data extraction on each of these articles.
Disagreements were resolved by consensus, and a third
reviewer was consulted when necessary. Table 2 lists the
items extracted.
We treated each method for determining MGMT pro-
moter methylation status as a separate prognostic factor
and extracted preferentially an unadjusted HR and its con-
fidence interval (CI) for each method. Where unadjusted

Downloaded from https://academic.oup.com/neuro-oncology/article/23/9/1457/6263562 by guest on 18 April 2023

  
5494 records identified through database searching 20 additional informative
articles from references to
Ovid MEDLINE (1946 to 4-Dec-2018), n = 1500
included studies
PubMed NOT MEDLINE (4-Dec-2018), n = 101
Ovid Embase (1980 to 2018, Week 49), n = 2983
BIOSIS (1969 to 3-Dec-2018), n = 790
CPCI-S (1900 to 3-Dec-2018), n = 120

3357 record after

duplicates removed

3357 record 1590 records excluded

screened

Records excluded = 1526

1767 full-text articles
assessed for eligibility
Full-text articles excluded, with reasons (n = 7)
[see characteristics of excluded studies table]
Other-1 (n = 846)
[Some studies excluded with multiple reasons]
Wrong study design (67)
Wrong population (453)
MGMT measurement (241)
Wrong outcome (258)
Wrong publication type (28)
Other-2 (n = 673)
Conference abstracts (no eligible data) (608)
Single technique: HR not repoeted/can’t be
calculated (49)
Linked to another paper (5)
Other (11)
+ Awaiting Classification, n = 17
+ Ongoing Studies, n = 1

190 articles providing

223 articles includes quantitative infotmation on
in the review studies of a single method

32 seperate cohorts
(described in (33 + 20)
articles) on which multiple
methods were evaluated
using hazard ratios
(included in the
quantitative analyses)

Fig. 1  Study flow diagram illustrating the selection process of records identified in the search.
  
 Brandner et al. Meta-analysis of the predictive value of MGMT promoter methylation 1461

Oncology
Neuro-
  
Table 2  Parameters Captured and Assessed for Each Included Study of 2 or More Methods

Study characteristics Author

Year
Country
Length of follow-up
Study dates
Study design
Population characteristics Number of participants

Downloaded from https://academic.oup.com/neuro-oncology/article/23/9/1457/6263562 by guest on 18 April 2023

Population source and setting
Timing of MGMT promoter methylation assessment
Inclusion/exclusion criteria
Tumor type
Age
Gender
Karnofsky performance status
Extent of resection
Treatment regimen
Length of time between neurosurgery and start of
treatment
IDH mutation status
First diagnosis or recurrent disease
Deaths during follow-up
Prevalence of MGMT promoter methylation (by each
technique
Method(s) of MGMT promoter methylation assess- Technique
ment
Tumor sample type (ie, FFPE or frozen tissue)
Region/CpGs analyzed (for PCR-based tests); anti-
body used (for immunohistochemistry
Cutoff/threshold used to determine MGMT promoter
methylation status (where relevant)
Outcome assessment Timepoint from which overall survival is measured
Missing data Number of participants with any missing data

Abbreviations: FFPE, formalin-fixed paraffin-embedded; IDH, isocitrate dehydrogenase; MGMT, O6-methylguanine-DNA methyltransferase.
HRs were not reported directly, we obtained them from re-
ported individual participant data (IPD), reported adjusted
HR’s or reconstructed IPD from published Kaplan-Meier
survival curves.47 When IPD or reconstructed IPD available
for 3 or more groups, the groups were combined to enable
2-way comparison (eg, by comparing “unmethylated” with
combined “weakly methylated” and “methylated”).
For studies that evaluated MGMT promoter methyla-
tion status using only a single method, a single reviewer
extracted information on author, year, country, follow-up,
number of participants, tumor type, IDH mutation status,
and MGMT technique.
Assessment of Risk of Bias
The risk of bias in studies evaluating MGMT promoter
methylation status of the same patients using at least 2
methods was assessed with a modified version of the
Quality in Prognosis Studies (QUIPS) tool,48 across the
domains: study participation, subsequent treatment, out-
come measurement, prognostic factor measurement,
study attrition, adjustment for other potential prognostic
factors (where relevant), and selective reporting.
Data Synthesis and Meta-Analysis
The prognostic ability of each individual method was quan-
tified using a HR for overall survival, presented with a 95%
CI. Comparisons of tests were restricted to those that could
be made on the same patients within the same study. The
directions of HRs were harmonized to reflect a better out-
come with a greater HR. Where 5 or more studies had com-
pared the same pair of techniques on the same patients,
we computed ratio of hazard ratios (RHR), and combined
these across studies using standard random-effects meta-
analysis methods.49 We evaluated certainty in the evidence
following the GRADE framework.50
1462 Brandner et al. Meta-analysis of the predictive value of MGMT promoter methylation

  
CpGs Technique Sym 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99
3
MS-RE-qPCR
3

1
MS-RE-qPCR
Beadarray 1
3

Almuqate 2018
1

2
MSP
Bead array
1 1 1 1 1

1
1 1 1
1

Bady 2012 (M-GBM)

Bead array
Bady 2012/Etcheverry 2010 (E-GBM)
2 2 2 2 2 2 2 2
2

2 PSQ 2 2 2 2 2

Methyl-Beaming
Barault 2015
15 15 15 15 15 15

PSQ
15
15 15 15 15 15 15 15

16 MSP 16 16 16 16 16 16 16 16 16

16

16
MSP
PSQ
16 16 16 16 16 16 16 16 16 16

Barbagallo 2014
16

14
PSQ
QF-IHC(AQUA)
14

Bell 2017
qMSP
Brigliadori 2016
14
12

12 PSQ 12 12 12 12 12 12 12 12

PSQ
Chai 2018 (7-site cohort)
12 12 12 12 12 12 12 12 12

17 PSQ 17 17 17 17

17 PSQ 17 17 17 17 17 17 17 17
17

17

17
PSQ
PSQ 17 17 17 17
17

17
17

17
17

17
17
Chai 2018 (8-site cohort)
17

17
PSQ
PSQ
17 17

17
17

17
17

17
17

17
18
Dahlrot 2018 (NS cohort)
18

18
DIF
PSQ 18 18 18 18 18
Dahlrot 2018 (RSD cohort)
DIF
Dunn 2009
18

19
18 PSQ 18 18 18 18 18

Downloaded from https://academic.oup.com/neuro-oncology/article/23/9/1457/6263562 by guest on 18 April 2023

PSQ
Felsberg 2009
19 19 19 19 19 19 19 19 19 19 19 19 19

19 PSQ 19 19 19 19 19 19 19 19 19 19 19 19
10

19 PSQ 19 19 19 19 19 19 19 19 19 19 19 19

19

19
PSQ
PSQ
19

19
19

19
19

19
19

19
19

19
19

19
19

19
19

19
19

19
19

19
19

19
19

19
20

Havik 2012/Johannessen 2018

19

10
PSQ
IHC
19 19 19 19 19 19 19 19 19 19 19 19
21

Hsu 2015/2017
mRNA
Karayan-Tapon 2010
10

13

10 MSP
MSP
Kim 2016
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20

20 MSP 20 20 20 20 20 20 20 20 20
22

20 PCR with HRM 20 20 20 20 20 20 20 20 20 20 20 20

20

20
PSQ
PSQ
20

20
20

20
20

20
20

20
20

20
9

Kristensen 2016
20

20
PSQ
PSQ
20

20
20

20
20

20
20

20
20

20
23

Lalezari 2013
Lattanzio 2015
20 PSQ 20 20 20 20 20

24

20 PSQ 20 20 20 20

PSQ
Lechapt-Zalcman2012
20 20 20 20 20

PSQ
6
20 20 20 20 20

20 PSQ 20 20 20 20

20

21
qMSP
IHC
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 4

McDonald 2013
21

21
MSP
PSQ
21

21
21

21
21

21
21

21
21 21 21 21 21
7

Melguizo 2012
qMSP
Nguyen 2015
21 21 21 21 21 21 21 21 21
8

13 IHC
MSP
Park 2011
13 13 13 13 13 13 13 13 13 13

11
13 PSQ 13

13 PSQ 13

13

13
PSQ
PSQ
13

13
26

Quillien 2012/2014 (test)

13

13
PSQ
PSQ 13 13 13 13
13

13
27

Quillien 2014 (validation)

qRT-PCR
Quillien 2016/2017
13
25

13 SQ-MSP 13 13 13 13 13 13 13 13 13

MSP
Thon 2017
22 22 22 22 22 22 22 22 22 22

29
22 PSQ 22 22 22 22 22 22 22

IHC
Yamashita 2018
9

9 PSQ 30

9 qMSP-PSQ
9

9
qMSP-PSQ
qMSP-PSQ
31

Yang 2012
23

23
IHC
MSP 23 23 23 23 23 23 23 23 23
32

Yoshioka 2018
23 PSQ 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23

MSP

Test Symbol
24 24 24 24 24 24 24 24 24 24

24 MSP 24 24 24 24 24 24 24 24 24

24 PSQ 24 24 24 24 24 24 24 24 24

24 PSQ 24 24 24 24 24 24 24 24 24

6
IHC
MSP 6 6 6 6 6 6 6 6
Beadarray
4 MSP 4 4 4 4 4

7
PSQ
IHC
4 4 4 4 4

DIF
7 MSP 7 7 7 7 7 7 7 7

8
FSQ-MS-PCR
FSQ-MS-PCR
8

8
8

8
8

8
8

8
8

8
8

8
8

8
8

8
8

8
FSQ-MS-PCR
11 MS-MLPA
11

11
MS-MLPA
MSP 11 11 11 11 11 11 11 11
IHC
25 PSQ 25 25 25 25 25

25

25
PSQ
PSQ
25

25
25

25
25

25
25

25
25

25
MethyI-Beaming
25 PSQ 25 25 25 25 25

25

25
PSQ
PSQ
25

25
25

25
25

25
25

25
25

25
MethylLight-MSP
25 PSQ 25 25 25 25 25

25

25
PSQ
PSQ
25

25
25

25
25

25
25

25
25

25
mRNA
25 PSQ 25 25 25 25 25

25

25
PSQ
PSQ
25

25
25

25
25

25
25

25
MS-MLPA
25
SQ-MSP 25 25 25 25 25 25 25 25 25

SQ-MSP
MSP
25 25 25 25 25 25 25 25 25 25

25
SQ-MSP 25 25 25 25 25 25 25 25 25

25
SQ-MSP 25 25 25 25 25 25 25 25 25

MS-RE-qPCR
25 SQ-MSP 25 25 25 25 25 25 25 25 25

26 IHC
26
MethylLight-MSP 26 26 26 26 26 26 26 26 26 26 26 26

PCR with HRM

26 MSP 26 26 26 26 26 26 26 26 26

26 PCR with HRM 26 26 26 26 26 26 26 26 26 26 26 26 26 26

26 PSQ 26

PSQ
26 PSQ 26

26 PSQ 26

26 PSQ 26

PSQ
QF-IHC(AQUA)
26 26

26 PSQ 26

26 PSQ 26

PSQ
qMSP
26 26

26 PSQ 26

26 PSQ 26

PSQ
qMSP-PSQ
26 26

26 PSQ 26

26 PSQ 26

PSQ
qRT-PCR
26 26

26 PSQ 26

26 PSQ 26

PSQ
SQ-MSP
26 26

26 PSQ 26

26 PSQ 26

26 PSQ 26

26 PSQ 26 26 26 26 26

26 PSQ 26 26 26 26 26

26 PSQ 26 26 26 26 26 26 26 26 26 26 26 26 26 26 26 26

26 PSQ 26 26 26 26 26

26 PSQ 26 26 26 26

26 PSQ 26 26 26 26 26

26 PSQ 26 26 26 26 26

26 PSQ 26 26 26 26 26

26 PSQ 26 26 26 26 26

26 PSQ 26 26 26 26 26

26 PSQ 26 26 26 26 26 26

26 PSQ 26 26 26 26 26

27 PSQ 27 27 27 27 27

27 PSQ 27 27 27 27 27

27 PSQ 27 27 27 27 27

29 MSP 29 29 29 29 29 29 29 29 29

29 PSQ 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29

30 MSP 30 30 30 30 30 30 30 30 30

30 PCR with HRM 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30

30 PCR with HRM 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30

30 PCR with HRM 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30

30 PCR with HRM 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30

30 PCR with HRM 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30

31 IHC
31 MSP 31 31 31 31 31 31 31 31 31

32 MSP 32 32 32 32 32 32 32 32 32

32 MSP 32 32 32 32 32 32 32 32 32

32 MSP 32 32 32 32 32 32 32 32 32

32 MSP 32 32 32 32 32 32 32 32 32

32 MSP 32 32 32 32 32 32 32 32 32

Fig. 2  Schematic overview of the CpG sites tested in the different publications. The first column is a color-coded representation of the authors,
which are shown in the inset on the right. The CpG sites are listed in numerical order, corresponding to the iterative positions relative to transcrip-
tion start. The corresponding sites, test methods, and thresholds are shown in detail in the Supplementary data. Each row represents a distinct
method and where applicable, different CpG sites or thresholds. Rows with blank cells (ie, no color-coded CpG sites) indicate that a method was
not PCR-based test or that CpG information is not available. For studies using PCR primers as described by Esteller et al.51 CpG site location is
based on Malley et al.52
  
 Brandner et al. Meta-analysis of the predictive value of MGMT promoter methylation 1463

Additional Analyses
The full Cochrane Review includes more details of the
methods and further analyses including adjusted HRs
(examining the prognostic value of tests in addition to age
and extent of resection) and sensitivity analyses. In addi-
tion, it collates information about the UK costs of the main
techniques and cost comparison ratios.12
Oncology
Neuro-
America (n  =  2), East Asia (n  =  8), Australia (n  =  1), or in
multiple countries (n = 2). Average patient age ranged from
44 to 64, with an overall male: female ratio of 1.5:1. The vast
majority were patients with glioblastomas, predominantly
undergoing total resection. Figure 2 illustrates the CpG
sites targeted in the studies. The Supplementary data pro-
vide a comprehensive overview of the data from all indi-
vidual comparison studies.

Findings: Comparisons of Different Techniques

Results

Downloaded from https://academic.oup.com/neuro-oncology/article/23/9/1457/6263562 by guest on 18 April 2023

Results of the Search
The search identified 5494 records, of which 223 were in-
cluded in the review (see Figure 1). These comprised 32
separate cohorts of patients (“studies”) where 2 or more
methods were compared, including studies comparing
different variants of the same technique. About 190 fur-
ther articles describing single-technique studies were
also included and are described in a separate section
below.
Characteristics of the Included Studies
The 32 studies included a total of 3474 participants. The
techniques investigated and the corresponding references
are listed in Table 1. All studies had a standard cohort de-
sign. Studies were undertaken in Europe (n  =  19), North
The 160 extracted HRs are reported in the Supplementary
data and summarized in Table 3. In all cases, the estimated
HR is above 1, indicating higher hazard of death in those
with unmethylated MGMT promoters. In the vast majority
of cases, the lower limit of a 95% CI for the HR is above 1,
confirming the prognostic value of MGMT promoter meth-
ylation status. When examining these results, we empha-
size that comparisons should only be made of different
methods within studies. HRs should not be compared
across studies because there are many (more substantial)
differences between these results than the choice of tech-
nique, tumor sample, CpG islands, or thresholds.
Meta-analysis of RHR (Table 3) shows that MSP (CpG
sites 76-80 and 84-87) is more prognostic than IHC (varying
thresholds) with RHR  =  1.31 (95% CI: 1.01-1.71). Since a
large majority of MSP studies had examined CpG sites
76-80 and 84-87,52 we were unable to compare alternative
CpG sites for MSP. We also found evidence that PSQ is

  
Table 3  Summary of Findings of Comparisons of Methods for Measuring MGMT Promoter Methylation Status

Technique 1 Technique 2 RHR (95% CI) Partici- Studies Certainty of Reason for Down Rating
pants Evidence
MSP IHC 1.31 (1.01-1.71) 913 7 Moderate Imprecision
PSQ IHC 1.36 (1.01-1.84) 871 5 Low Imprecision and indirectness (due to variability in
CpG sites and thresholds used for PSQ)
PSQ MSP 1.14 (0.87-1.48) 1119 9 Low Imprecision and indirectness (due to variability in
CpG sites and thresholds used for PSQ)
PSQ PSQ (variant Not estimated 876 11 Very low Serious risk of bias, imprecision, inconsistency,
of) and indirectness
qMSP MSP of PSQ Not estimated 765 7 Very low Risk of bias, imprecision, inconsistency, and indi-
rectness
Bead array MSP of PSQ Not estimated 81 2 Very low Serious imprecision, inconsistency, and indirect-
ness
PCR-mRNA MSP or PSQ Not estimated 148 2 Very low Imprecision, inconsistency, and indirectness
MS-MLPA MSP or PSQ Not estimated 48 1 Very low Serious risk of bias, serious imprecision, incon-
sistency, and indirectness
PCR-HRM MSP or PSQ Not estimated 309 3 Very low Risk of bias, serious imprecision, inconsistency,
and indirectness
Others MSP or PSQ Not estimated 1209 7 Very low Serious imprecision, inconsistency, and indirect-
ness

Abbreviations: CI, confidence interval; RHR, ratio of hazard ratios; for technique abbreviations, see Table 2.
The outcome being predicted is overall mortality (time to death). Grades of evidence: high quality, further research is very unlikely to change our con-
fidence in the conclusion; moderate quality, further research is likely to have an important impact on our confidence in the conclusion; low quality,
further research is very likely to have an important impact on our confidence in the conclusion; very low quality, we are very uncertain about the
conclusion.
  
1464 Brandner et al. Meta-analysis of the predictive value of MGMT promoter methylation

more prognostic than IHC (RHR = 1.36; 95% CI: 1.01-1.84),
although studies of PSQ feeding into this analysis had tar-
geted different CpG sites. While there is a consistent pat-
tern that PSQ seems to be a slightly better predictor than
MSP, there is no strong statistical evidence to confirm this
(RHR = 1.14; 95% CI: 0.87-1.48). The CpG sites targeted by
PSQ ranged between 72 and 95, and several studies had
examined sites 74-78. There was a suggestion that PSQ
(mainly at CpG sites 74-78, but with varying thresholds) is
slightly more prognostic than MSP (sites 76-80 and 84-87).

  
Risk of bias
Colour code Study ID (author/year) Participant Subsequent Outcome
selection treatment measurement

Downloaded from https://academic.oup.com/neuro-oncology/article/23/9/1457/6263562 by guest on 18 April 2023

Almuqate 2018

Bady 2012 (M-GBM)

Bady 2012/Etcheverry 2010 (E-GBM)

Barault 2015

Barbagallo 2014

Bell 2017

Brigliadori 2016

Chai 2018 (7-site cohort)

Chai 2018 (8-site cohort)

Dahlrot 2018 (NS cohort)

Dahlrot 2018 (RSD cohort)

Dunn 2009

Felsberg 2009

Havik 2012/Johannessen 2018

Hsu 2015/2017

Karayan-Tapon 2010

Kim 2016

Kristensen 2016

Lalezari 2013

Lattanzio 2015

Lechapt-Zalcman 2012

McDonald 2013

Melguizo 2012

Nguyen 2015

Park 2011

Quillien 2012/2014 (test)

Quillien 2014 (validation)

Quillien 2016/2017

Thon 2017

Yamashita 2018

Yang 2012

Yoshioka 2018

Fig. 3  Study-level risk-of-bias assessments for studies comparing 2 or more methods. participant selection, subsequent treatment, and out-
come. Green (+) = low risk of bias; Yellow (−) = unclear risk of bias. The color codes of the individual studies correspond to those in Figure 1.
Abbreviations: GBM, glioblastoma multiforme; NS, Nordic Study; RSD, Region of Southern Denmark.
  
 Brandner et al. Meta-analysis of the predictive value of MGMT promoter methylation 1465

We did not perform formal analyses to investigate
whether heterogeneity in HRs may have been due to age,
extent of tumor resection, Karnofsky performance status,
IDH status, first diagnosis vs recurrence, start and length
of follow-up, due to the very limited replication of specific
methods, and large amounts of missing data for many of
these study characteristics.
Many variants of PSQ have been compared, although we
did not see any strong and consistent messages from the
results. Thresholds varied substantially (from 4% to 25%
for single CpG sites; and from 2.68% to 35% for multiple
CpG sites). Two of the three studies with low (or unclear)
Oncology
Neuro-
Risk-of-Bias Assessment and Certainty in the
Evidence
We present results of the risk-of-bias assessment for the
3 domains that apply to the whole studies in Figure 3. All
studies were assessed to be at low or unclear risk of bias for
participant selection. All studies except one were assessed
as at low risk of bias arising due to variation in subsequent
treatment after collection of the tumor sample. All studies
were assessed to be at low risk of bias in measurement of
the outcome (all-cause mortality). The other aspects of the
risk-of-bias assessment apply to individual results. We were

Downloaded from https://academic.oup.com/neuro-oncology/article/23/9/1457/6263562 by guest on 18 April 2023

risk of bias that compared different thresholds found that a
9% threshold was more prognostic than higher thresholds
(of 28% or 29%; see top 2 results in Figure 4). We are un-
able to draw strong conclusions about use of frozen tissue
vs FFPE in MSP, although 1 study observed that MSP was
more prognostic when based on frozen tissue. No clear
difference was apparent between using PSQ on FFPE vs
frozen tissue.
mostly free of concerns about risk of bias in the domains
for study attrition, problems with other prognostic factors
adjusted for, and selective reporting. For some results, the
threshold used to classify methylation status was derived
from the data, leading to a high risk of bias. The result-level
risk-of-bias assessments for studies examining PSQ are in-
cluded in Figure 4. Table 3 summarizes the certainty of the
evidence from comparative studies, grouped by technique.

  
Table 4  Characteristics of Studies Examining MGMT Promoter Methylation With 1 Technique Only

Study Parameter Characteristics No. of Studies

Total number of studies 190
Reporting follow-up information 54
Reporting follow-up range 29
Reporting data on IDH1/IDH2 mutation 62
All IDH wild type 11
IDH mutation present (0.7%-73.4%) 47
No IDH mutation reported 3
Reporting tumor type Glioblastomas only (all studies) 183
Glioblastoma: supratentorial 9
Glioblastoma: primary 23
Glioblastoma: primary, supratentorial 1
Glioblastoma: recurrent 4
Mixed glioma + gliosarcoma 6
Gliosarcoma only 1
Test method MSP 94
PSQ 27
qMSP (real-time PCR or MethyLight) 22
Bead array 10
MS-MLPA 4
HRM-PCR 3
MGMT protein (IHC) 21
MGMT protein (Western blot) 1
mRNA 4

Abbreviations: HRM-PCR, PCR with high-resolution melting; IDH, isocitrate dehydrogenase; IHC, immunohistochemistry; MGMT, O6-
methylguanine-DNA methyltransferase; MS-MLPA, methylation-specific multiplex ligation-dependent probe amplification; MSP, methylation-specific
PCR; PSQ, pyrosequencing; qMSP, quantitative methylation-specific PCR.
As per the study protocol, the results of these studies were not examined, because comparisons of HRs across studies would not provide reliable
indicators of differences between the methods.
  
1466 Brandner et al. Meta-analysis of the predictive value of MGMT promoter methylation

  
Study ID and CpGsanalysed Threshold for Hazard ratio PF Selective
Sample type (PCR-based tests) methylated (95% CI) measurement Attrition reporting
Barbagallo 2014
FFPE NR >9% 3.73 (1.68, 8.28) Low RoB Low RoB Low RoB
FFPE NR >25% 1.99 (0.92, 4.32) Low RoB Low RoB Low RoB

Brigliadori 2016
FFPE 74-83 >9% 1.92 (1.17, 3.14) Low RoB Low RoB Low RoB
FFPE 74-83 >29% 3.02 (1.72, 5.29) Low RoB Low RoB Low RoB

Chai 2018 (7-site cohort)

Frozen 72-78 >12% 2.94 (1.12, 7.69) High RoB Low RoB Unclear
Frozen 74-78 >12% 2.94 (1.12, 7.69) High RoB Low RoB Unclear
Frozen 75-78 >12% 2.94 (1.12, 7.69) High RoB Low RoB Unclear

Chai 2018 (8-site cohort)

Frozen 75-78 >13% 2.70 (1.37, 5.26) High RoB Low RoB Unclear
Frozen 75-82 >12% 3.03 (1.54, 6.25) High RoB Low RoB Unclear
Frozen 76-79 >11% 2.13 (1.09, 4.17) High RoB Low RoB Unclear

Downloaded from https://academic.oup.com/neuro-oncology/article/23/9/1457/6263562 by guest on 18 April 2023

Dunn 2009
Frozen, smear and/or FFPE 72-83 >9% 3.57 (2.24, 5.70) Low RoB Low RoB Low RoB
Frozen, smear and/or FFPE 72-83 >20% 4.25 (2.57, 7.05) Unclear Low RoB Low RoB
Frozen, smear and/or FFPE 72-83 >29% 4.03 (2.30, 7.07) High RoB Low RoB Low RoB
Frozen, smear and/or FFPE 72-83 >35% 3.64 (1.99, 6.67) Unclear Low RoB Low RoB
Frozen, smear and/or FFPE 72-83 Cluster 1 vs 2&3 4.44 (2.58, 7.66) Unclear Low RoB Low RoB
Frozen, smear and/or FFPE 72-83 Cluster 1&2 vs 3 3.59 (2.26, 5.69) Unclear Low RoB Low RoB

Havik 2012
Frozen 74-78 >2.68% 1.85 (1.02, 3.35) Low RoB Low RoB Low RoB
Frozen 74-78 >6% 2.30 (1.21, 4.38) High RoB Low RoB Low RoB
Frozen 74-78 >7% 2.33 (1.19, 4.57) High RoB Low RoB Low RoB
Frozen 74-78 >8% 2.33 (1.19, 4.57) High RoB Low RoB Low RoB
Frozen 74-78 >9% 2.30 (0.99, 5.33) High RoB Low RoB Low RoB
Frozen 76-79 >6% 2.30 (1.21, 4.38) High RoB Low RoB Low RoB
Frozen 76-79 >7% 2.33 (1.19, 4.57) High RoB Low RoB Low RoB
Frozen 76-79 >8% 1.90 (0.99, 3.65) High RoB Low RoB Low RoB
Frozen 76-79 >9% 1.90 (0.99, 3.65) High RoB Low RoB Low RoB

Karayan-Tapon 2010
Frozen 74 >5.5% 3.26 (1.91, 5.59) Low RoB Low RoB Low RoB
Frozen 74-78 >8.0% 3.35 (1.95, 5.73) Low RoB Low RoB Low RoB
Frozen 75 >8.7% 3.26 (1.91, 5.57) Low RoB Low RoB Low RoB
Frozen 76 >8.0% 2.78 (1.64, 4.74) Low RoB Low RoB Low RoB
Frozen 77 >7.85% 3.65 (2.10, 6.34) Low RoB Low RoB Low RoB
Frozen 78 >7.8% 2.70 (1.59, 4.58) Low RoB Low RoB Low RoB

Lattanzio 2015
FFPE 72-80 ?9% 2.09 (1.09, 3.99) Low RoB Low RoB Low RoB
Frozen 72-80 ?9% 2.25 (1.19, 4.25) Low RoB Low RoB Low RoB

Quillien 2014 (test)

Frozen 74 >4% 2.44 (1.51, 3.95) High RoB Low RoB Low RoB
Frozen 74 >8% 2.22 (1.30, 3.79) High RoB Low RoB Low RoB
Frozen 74-78 >8% 3.13 (1.86, 5.25) High RoB Low RoB Low RoB
Frozen 74-78 >9% 3.13 (1.81, 5.38) High RoB Low RoB Low RoB
Frozen 74-89 >11% 3.13 (1.84, 5.30) High RoB Low RoB Low RoB
Frozen 75 >11% 3.23 (1.87, 5.55) High RoB Low RoB Low RoB
Frozen 75-79 >8% 2.94 (1.71, 5.06) High RoB Low RoB Low RoB
Frozen 76 >4% 2.63 (1.61, 4.31) High RoB Low RoB Low RoB
Frozen 76 >5% 2.78 (1.62, 4.75) High RoB Low RoB Low RoB
Frozen 76-79 >8% 2.86 (1.66, 4.92) High RoB Low RoB Low RoB
Frozen 76-80 >9% 3.03 (1.74, 5.26) High RoB Low RoB Low RoB
Frozen 77 >6% 3.13 (1.87, 5.23) High RoB Low RoB Low RoB
Frozen 77 >7% 2.78 (1.63, 4.74) High RoB Low RoB Low RoB
Frozen 77-81 >8% 2.70 (1.59, 4.59) High RoB Low RoB Low RoB
Frozen 78 >4% 2.33 (1.38, 3.92) High RoB Low RoB Low RoB
Frozen 78 >5% 2.50 (1.51, 4.13) High RoB Low RoB Low RoB
Frozen 78-82 >9% 2.86 (1.68, 4.86) High RoB Low RoB Low RoB
Frozen 79 >7% 2.78 (1.61, 4.80) High RoB Low RoB Low RoB
Frozen 79-83 >8% 2.86 (1.68, 4.86) High RoB Low RoB Low RoB
Frozen 80 >4% 2.56 (1.54, 4.28) High RoB Low RoB Low RoB
Frozen 81 >8% 2.44 (1.44, 4.14) High RoB Low RoB Low RoB
Frozen 82 >16% 2.94 (1.70, 5.08) High RoB Low RoB Low RoB
Frozen 83 >10% 2.78 (1.65, 4.66) High RoB Low RoB Low RoB
Frozen 84 >9% 3.23 (1.86, 5.59) High RoB Low RoB Low RoB
Frozen 84-88 >17% 3.23 (1.86, 5.58) High RoB Low RoB Low RoB
Frozen 84-89 >22% 3.23 (1.85, 5.62) High RoB Low RoB Low RoB
Frozen 85 >5% 2.50 (1.52, 4.11) High RoB Low RoB Low RoB
Frozen 85-89 >13% 2.94 (1.76, 4.92) High RoB Low RoB Low RoB
Frozen 86 >11% 2.78 (1.65, 4.69) High RoB Low RoB Low RoB
Frozen 87 >25% 3.03 (1.75, 5.24) High RoB Low RoB Low RoB
Frozen 88 >4% 2.27 (1.38, 3.75) High RoB Low RoB Low RoB
Frozen 89 >12% 3.23 (1.91, 5.46) High RoB Low RoB Low RoB

Quillien 2014 (validation)

FFPE 74-78 >9% 3.70 (1.71, 8.01) Low RoB Low RoB Low RoB
FFPE 74-78 >10% 2.86 (1.42, 5.74) Low RoB Low RoB Low RoB
FFPE 74-78 >28% 2.27 (0.98, 5.29) Low RoB Low RoB Low RoB

Quillien 2016
FFPE 74-78 >6% 3.23 (2.02, 5.16) High RoB Unclear Low RoB
FFPE 74-78 >8% 4.00 (2.30, 6.97) Low RoB Unclear Low RoB
FFPE 74-78 >12% 4.17 (2.35, 7.38) Unclear Unclear Low RoB
FFPE 74-78 >13% 4.35 (2.41, 7.83) Unclear Unclear Low RoB
FFPE 74-78 >16% 4.55 (2.48, 8.34) High RoB Unclear Low RoB
Frozen 74-78 >6% 4.00 (2.30, 6.97) High RoB Unclear Low RoB
Frozen 74-78 >8% 3.57 (2.14, 5.95) Low RoB Unclear Low RoB
Frozen 74-78 >12 or 13% 3.45 (2.10, 5.66) Unclear Unclear Low RoB
Frozen 74-78 >12% 3.70 (2.19, 6.26) Low RoB Unclear Low RoB
Frozen 74-78 >16% 3.13 (1.94, 5.04) High RoB Unclear Low RoB
Frozen 76-79 >8% 3.33 (2.06, 5.40) Low RoB Unclear Low RoB
Frozen 76-79 >12% 3.33 (2.06, 5.40) Low RoB Unclear Low RoB

.5 1 2 4

Fig. 4  Hazard ratios from studies comparing different methods for PSQ. Hazard ratios from studies comparing different methods for PSQ. The
scale on the bottom of the figure indicates the hazard ratio. Abbreviations: CI, confidence interval; CpG, 5′-cytosine-phosphate-guanine-3′; FFPE,
formalin-fixed paraffin-embedded; NR, not reported; PF, prognostic factor; PSQ, pyrosequencing; RoB, risk of bias.
  
 Brandner et al. Meta-analysis of the predictive value of MGMT promoter methylation
Studies Examining Only a Single Technique
About 190 articles described studies presenting HRs from
survival analysis in patients in which MGMT methylation
was measured by 1 technique, and studies in which more
than 1 technique was used but only MGMT methylation data
from 1 method were used in the survival analysis (Table 4).
These studies included a total of 27 710 participants (range
6-1395). They were conducted in Italy (n = 29), multiple coun-
tries (n = 23), Germany (n = 21), the United States (n = 20),
Japan (n = 18), China (n = 17), South Korea (n = 11), France
(n = 9), Denmark (n = 8), Spain (n = 8), the United Kingdom
(n = 6), India (n = 3), Switzerland (n = 3), Australia, Belgium,
Czech Republic, Egypt, Taiwan (n = 2), and 1 study each in
Canada, Portugal, Netherlands, and Tunisia.
Discussion
We took a systematic approach to identifying, appraising,
and collecting information from the evidence and assessed
risk of bias and applicability concerns using a modification
of QUIPS specific to the topic of the Cochrane Review.48
This is the first systematic review to our knowledge that
compares methods for categorizing tumors as methylated
in relation to their ability to predict survival in patients
with glioblastoma. Unsurprisingly, among methods for
assessing MGMT status in glioblastoma patients treated
with temozolomide, PSQ and MSP appear to be more
prognostic for overall survival than IHC. While there is a
consistent pattern that PSQ seems to be a slightly better
predictor than MSP, there is no strong statistical evidence
to confirm this. Moreover, there is no strong evidence to
draw conclusions with confidence about the best CpG sites
or thresholds for quantitative methods. In our study, MSP
has been studied mainly for CpG sites 76-80 and 84-87 and
PSQ at CpG sites ranging from 72 to 95. A cutoff threshold
of 9% for CpG sites 74-78 was found to perform better than
higher thresholds of 28% or 29% in 2 of the 3 good quality
studies making such comparisons.13,14,53
To ensure fair comparison of methods, we assessed
comparisons on the same patients and tumors within a
study. A large variety of variants have been examined, par-
ticularly the use of different CpG sites and thresholds for
PSQ, as well as a mixture of use of FFPE and frozen tumor
samples. There was only a small amount of direct replica-
bility across studies, meaning that firm conclusions were
difficult to draw.
We limited eligibility for the review to studies that re-
ported HRs or data sufficient for us to estimate them. In
many instances, we reconstructed time-to-event data from
Kaplan-Meier curves, allowing us to include 14 studies that
we would not have included otherwise. However, there
were still a small number of studies that had sought to
compare methods but not presented data compatible with
computation of HRs, which therefore did not meet our eli-
gibility criteria.
We listed brief details of articles describing studies
examining only 1 technique in the full Cochrane Review,
although these were not included in the final meta-
analysis (Table 4 and reference 12). Among the studies
that compared multiple techniques, we observed that HRs
1467
Oncology
Neuro-
varied markedly across studies, and we were unwilling to
make naive indirect comparisons of techniques across dif-
ferent studies and we are presenting quantitative results
for these studies.12
We rated the evidence for the comparison between MSP
and IHC as of “moderate certainty,” and the evidence for
comparisons of PSQ with MSP or IHC as of “low certainty”
(Table 3). All other comparisons we rated as “very low cer-
tainty.” Although risk-of-bias and publication bias were not
major concerns for us, we rated down many of our assess-
ments because there was a wide variety of different CpG
sites and thresholds investigated, without systematic repli-
Downloaded from https://academic.oup.com/neuro-oncology/article/23/9/1457/6263562 by guest on 18 April 2023
cations of findings using the same methods across studies.
The amount of evidence is small, with only tens or at most
the low hundreds of participants contributing to evidence
for many of the techniques.
The evidence identified was generally applicable to clinical
practice. We included only studies in which at least 90% of pa-
tients had glioblastoma, and nearly all patients were treated
with temozolomide. We focused on overall survival only, so
are unable to draw conclusions about using techniques to
predict progression-free survival. The decision which method
to use in clinical practice however is not necessarily guided
by best predictive value but is influenced by cost, turnaround
time, availability of equipment: PSQ, the most quantitative
method can be limited by the availability of equipment, while
qMSP, a commonly used method, cannot accurately quantify
heterogeneously methylated CpG sites.
Further large studies examining the use of different tech-
niques, using pre-defined threshold values for interpre-
tation, would provide valuable new information on these
methods, and our review reflects the reality that it may be
challenging to reach a consensus for the best method of
MGMT promoter methylation testing.
Supplementary Material
Supplementary material is available at Neuro-Oncology
online.
Keywords
glioblastoma | meta-analysis | MGMT promoter methyla-
tion | prognostic biomarker | temozolomide
Acknowledgments
We thank Robin Grant (Co-ordinating Editor) and Gail Quinn
(Managing Editor) for the Cochrane Gynaecological, Neuro-
oncology and Orphan Cancers Group (GNOC) for their editorial
guidance and patience. We also thank Joanne Platt (Information
Specialist, GNOC) for aiding with the development of the search
strategy. We also thank all of our external peer reviewers, in-
cluding Karel Moons and Joie Ensor from the Cochrane
Prognostic Methods Group, Catherine McBain, Andrew Bryant,
Helen Bulbeck, and Colin Smith.
1468 Brandner et al. Meta-analysis of the predictive value of MGMT promoter methylation
Funding
This project was supported in part by the National Institute for
Health Research (NIHR), via Cochrane Programme Grant funding
(16/144) to the Cochrane Gynaecological, Neuro-oncology and
Orphan Cancers Group. A.M., F.S., and J.P.T.H. were supported in
part by Cancer Research United Kingdom (grant number C18281/
A19169). S.B.  was partly supported by the NIHR Biomedical
Research Centre to University College London Hospitals.
Conflict of interest statement. The views and opinions expressed
herein are those of the review authors and do not necessarily
reflect those of the NIHR Systematic Reviews Programme, the
NIHR, the National Health Service (NHS), the Department of
Health, or Cancer Research UK.
Authorship statement. A.M., J.P.T.H., and K.M.K.  wrote the
protocol; S.D. designed and undertook the searches; K.M.K.,
C.L.F., S.B., and C.W.  provided a clinical perspective; A.M.,
and K.M.K. screened search results; A.M., C.K., F.S., K.M.K.,
S.B., and C.L.F.  screened retrieved papers against eligibility
criteria; A.M., C.K., F.S., L.S., H.C., and J.P.T.H.  extracted
data from papers; A.M., C.K., F.S., L.S., and H.C.  appraised
quality of included studies; J.P.T.H.  and H.C.  undertook the
statistical analyses; J.P.T.H. undertook GRADE assessments;
K.M.K., S.B., J.P.T.H., F.S., and C.L.F.  interpreted the results;
J.P.T.H.  and K.M.K.  co-ordinated the review; J.P.T.H.  wrote
up the full Cochrane Review with input from all authors; S.B.,
K.M.K., and J.P.T.H. wrote this summary version.
References
1. Louis  DN, Perry  A, Reifenberger  G, et  al. The 2016 World Health
Organization classification of tumors of the central nervous system: a
summary. Acta Neuropathol. 2016;131(6):803–820.
2. Esteller M, Garcia-Foncillas J, Andion E, et al. Inactivation of the DNA-
repair gene MGMT and the clinical response of gliomas to alkylating
agents. N Engl J Med. 2000;343(19):1350–1354.
3. Malmström  A, Grønberg  BH, Marosi  C, et  al.; Nordic Clinical Brain
Tumour Study Group (NCBTSG). Temozolomide versus standard 6-week
radiotherapy versus hypofractionated radiotherapy in patients older
than 60 years with glioblastoma: the Nordic randomised, phase 3 trial.
Lancet Oncol. 2012;13(9):916–926.
4. Wick  W, Platten  M, Meisner  C, et  al.; NOA-08 Study Group of
Neuro-oncology Working Group (NOA) of German Cancer Society.
Temozolomide chemotherapy alone versus radiotherapy alone for malig-
nant astrocytoma in the elderly: the NOA-08 randomised, phase 3 trial.
Lancet Oncol. 2012;13(7):707–715.
5. Weller M, van den Bent M, Tonn JC, et al.; European Association for
Neuro-Oncology (EANO) Task Force on Gliomas. European Association
for Neuro-Oncology (EANO) guideline on the diagnosis and treat-
ment of adult astrocytic and oligodendroglial gliomas. Lancet Oncol.
2017;18(6):e315–e329.
6. National Institute for Health and Care Excellence (NICE). Brain tumours
(primary) and brain metastases in adults [NG99]. https://www.nice.org.
uk/guidance/ng99. Accessed June 2021.
7. Hegi ME, Stupp R. Withholding temozolomide in glioblastoma patients
with unmethylated MGMT promoter—still a dilemma? Neuro Oncol.
2015;17(11):1425–1427.
8. Brandner S, von Deimling A. Diagnostic, prognostic and predictive rel-
evance of molecular markers in gliomas. Neuropathol Appl Neurobiol.
2015;41(6):694–720.
9. Hegi  ME, Diserens  AC, Gorlia  T, et  al. MGMT gene silencing
and benefit from temozolomide in glioblastoma. N Engl J Med.
2005;352(10):997–1003.
Downloaded from https://academic.oup.com/neuro-oncology/article/23/9/1457/6263562 by guest on 18 April 2023
10. Bady P, Sciuscio D, Diserens AC, et al. MGMT methylation analysis of
glioblastoma on the Infinium methylation BeadChip identifies two dis-
tinct CpG regions associated with gene silencing and outcome, yielding
a prediction model for comparisons across datasets, tumor grades, and
CIMP-status. Acta Neuropathol. 2012;124(4):547–560.
11. Capper  D, Jones  DTW, Sill  M, et  al. DNA methylation-based
classification of central nervous system tumours. Nature.
2018;555(7697):469–474.
12. McAleenan A, Kelly C, Spiga F, et al. Prognostic value of test(s) for O6-
methylguanine-DNA methyltransferase (MGMT) promoter methylation
for predicting overall survival in people with glioblastoma treated with
temozolomide. Cochrane Database Syst Rev. 2021;3:CD013316.
13. Quillien V, Lavenu A, Sanson M, et al. Outcome-based determination of
optimal pyrosequencing assay for MGMT methylation detection in glio-
blastoma patients. J Neurooncol. 2014;116(3):487–496.
14. Dunn J, Baborie A, Alam F, et al. Extent of MGMT promoter methylation
correlates with outcome in glioblastomas given temozolomide and radi-
otherapy. Br J Cancer. 2009;101(1):124–131.
15. Dahlrot  RH, Dowsett  J, Fosmark  S, et  al. Prognostic value of O-6-
methylguanine-DNA methyltransferase (MGMT) protein expression in
glioblastoma excluding nontumour cells from the analysis. Neuropathol
Appl Neurobiol. 2018;44(2):172–184.
16. Håvik  AB, Brandal  P, Honne  H, et  al. MGMT promoter methylation in
gliomas-assessment by pyrosequencing and quantitative methylation-
specific PCR. J Transl Med. 2012;10:36.
17. Johannessen  LE, Brandal  P, Myklebust  TÅ, Heim  S, Micci  F,
Panagopoulos  I. MGMT gene promoter methylation status - assess-
ment of two pyrosequencing kits and three methylation-specific PCR
methods for their predictive capacity in glioblastomas. Cancer Genomics
Proteomics. 2018;15(6):437–446.
18. Hsu  CY, Ho  HL, Lin  SC, et  al. Prognosis of glioblastoma with
faint MGMT methylation-specific PCR product. J Neurooncol.
2015;122(1):179–188.
19. Hsu CY, Ho HL, Lin SC, et al. Comparative assessment of 4 methods to
analyze MGMT status in a series of 121 glioblastoma patients. Appl
Immunohistochem Mol Morphol. 2017;25(7):497–504.
20. Karayan-Tapon  L, Quillien  V, Guilhot  J, et  al. Prognostic value
of O6-methylguanine-DNA methyltransferase status in glioblas-
toma patients, assessed by five different methods. J Neurooncol.
2010;97(3):311–322.
21. Kim DC, Kim KU, Kim YZ. Prognostic role of methylation status of the
MGMT promoter determined quantitatively by pyrosequencing in glio-
blastoma patients. J Korean Neurosurg Soc. 2016;59(1):26–36.
22. Kristensen LS, Michaelsen SR, Dyrbye H, et al. Assessment of quanti-
tative and allelic MGMT methylation patterns as a prognostic marker in
glioblastoma. J Neuropathol Exp Neurol. 2016;75(3):246–255.
23. McDonald KL, Rapkins RW, Olivier J, et al. The T genotype of the MGMT
C>T (rs16906252) enhancer single-nucleotide polymorphism (SNP) is as-
sociated with promoter methylation and longer survival in glioblastoma
patients. Eur J Cancer. 2013;49(2):360–368.
 Brandner et al. Meta-analysis of the predictive value of MGMT promoter methylation
24. Quillien V, Lavenu A, Ducray F, et al. Validation of the high-performance
of pyrosequencing for clinical MGMT testing on a cohort of glioblastoma
patients from a prospective dedicated multicentric trial. Oncotarget.
2016;7(38):61916–61929.
25. Quillien  V, Lavenu  A, Ducray  F, et  al. Clinical validation of the CE-IVD
marked Therascreen MGMT kit in a cohort of glioblastoma patients.
Cancer Biomark. 2017;20(4):435–441.
26. Quillien  V, Lavenu  A, Karayan-Tapon  L, et  al. Comparative assess-
ment of 5 methods (methylation-specific polymerase chain reaction,
MethyLight, pyrosequencing, methylation-sensitive high-resolution
melting, and immunohistochemistry) to analyze O6-methylguanine-
DNA-methyltranferase in a series of 100 glioblastoma patients. Cancer.
2012;118(17):4201–4211.
27. Thon N, Thorsteinsdottir J, Eigenbrod S, et al. Outcome in unresectable
glioblastoma: MGMT promoter methylation makes the difference. J
Neurol. 2017;264(2):350–358.
28. Barbagallo GM, Paratore S, Caltabiano R, et al. Long-term therapy with
temozolomide is a feasible option for newly diagnosed glioblastoma: a
single-institution experience with as many as 101 temozolomide cycles.
Neurosurg Focus. 2014;37(6):E4.
29. Bell  EH, Pugh  SL, McElroy  JP, et  al. Molecular-based recursive
partitioning analysis model for glioblastoma in the temozolomide era: a
correlative analysis based on NRG oncology RTOG 0525. JAMA Oncol.
2017;3(6):784–792.
30. Felsberg  J, Thon  N, Eigenbrod  S, et  al.; German Glioma Network.
Promoter methylation and expression of MGMT and the DNA mismatch
repair genes MLH1, MSH2, MSH6 and PMS2 in paired primary and re-
current glioblastomas. Int J Cancer. 2011;129(3):659–670.
31. Lalezari  S, Chou  AP, Tran  A, et  al. Combined analysis of O6-
methylguanine-DNA methyltransferase protein expression and promoter
methylation provides optimized prognostication of glioblastoma out-
come. Neuro Oncol. 2013;15(3):370–381.
32. Lattanzio L, Borgognone M, Mocellini C, et al. MGMT promoter methyla-
tion and glioblastoma: a comparison of analytical methods and of tumor
specimens. Int J Biol Markers. 2015;30(2):e208–e216.
33. Melguizo C, Prados J, González B, et al. MGMT promoter methylation
status and MGMT and CD133 immunohistochemical expression as prog-
nostic markers in glioblastoma patients treated with temozolomide plus
radiotherapy. J Transl Med. 2012;10:250.
34. Park CK, Kim J, Yim SY, et al. Usefulness of MS-MLPA for detection of
MGMT promoter methylation in the evaluation of pseudoprogression in
glioblastoma patients. Neuro Oncol. 2011;13(2):195–202.
35. Yamashita S, Yokogami K, Matsumoto F, et al. MGMT promoter meth-
ylation in patients with glioblastoma: is methylation-sensitive high-
resolution melting superior to methylation-sensitive polymerase chain
reaction assay? J Neurosurg. 2018;130(3):780–788.
36. Yang  SH, Lee  KS, Yang  HJ, et  al. O6-methylguanine-DNA-
methyltransferase promoter methylation assessment by microdissection-
assisted methylation-specific PCR and high resolution melting analysis in
patients with glioblastomas. J Neurooncol. 2012;106(2):243–250.
37. Yoshioka M, Matsutani T, Hara A, et al. Real-time methylation-specific
PCR for the evaluation of methylation status of MGMT gene in glioblas-
toma. Oncotarget. 2018;9(45):27728–27735.
38. Felsberg J, Rapp M, Loeser S, et al. Prognostic significance of molecular
markers and extent of resection in primary glioblastoma patients. Clin
Cancer Res. 2009;15(21):6683–6693.
1469
Oncology
Neuro-
39. Lechapt-Zalcman E, Levallet G, Dugué AE, et al. O6-methylguanine-DNA
methyltransferase (MGMT) promoter methylation and low MGMT-
encoded protein expression as prognostic markers in glioblastoma
patients treated with biodegradable carmustine wafer implants after
initial surgery followed by radiotherapy with concomitant and adjuvant
temozolomide. Cancer. 2012;118(18):4545–4554.
40. Nguyen  A, Legrain  M, Noel  G, et  al. An innovative fluorescent semi-
quantitative methylation-specific PCR method for the determination of
MGMT promoter methylation is reflecting intra-tumor heterogeneity.
Curr Cancer Drug Targets. 2015;15(7):624–640.
41. Bady  P, Sciuscio  D, Stupp  R, Delorenzi  M, Hegi  ME, Grp  EBT. MGMT
methylation based outcome prediction is associated with two CpG re-
Downloaded from https://academic.oup.com/neuro-oncology/article/23/9/1457/6263562 by guest on 18 April 2023
gions separated by a prediction minimum centred at the initiation start
site. Cancer Res. 2012;72(Suppl. 8):4031.
42. Barault  L, Amatu  A, Bleeker  FE, et  al. Digital PCR quantification of
MGMT methylation refines prediction of clinical benefit from alkylating
agents in glioblastoma and metastatic colorectal cancer. Ann Oncol.
2015;26(9):1994–1999.
43. Binabaj MM, Bahrami A, ShahidSales S, et al. The prognostic value of
MGMT promoter methylation in glioblastoma: a meta-analysis of clin-
ical trials. J Cell Physiol. 2018;233(1):378–386.
44. Zhao H, Wang S, Song C, Zha Y, Li L. The prognostic value of MGMT
promoter status by pyrosequencing assay for glioblastoma patients’ sur-
vival: a meta-analysis. World J Surg Oncol. 2016;14(1):261.
45. Zhang K, Wang XQ, Zhou B, Zhang L. The prognostic value of MGMT
promoter methylation in glioblastoma multiforme: a meta-analysis. Fam
Cancer. 2013;12(3):449–458.
46. EPPI-Reviewer: advanced software for systematic reviews, maps and
evidence synthesis [computer program]. London: EPPI-Centre Software
UCL Social Research Institute; 2020.
47. Guyot P, Ades AE, Ouwens MJ, Welton NJ. Enhanced secondary
analysis of survival data: reconstructing the data from pub-
lished Kaplan-Meier survival curves. BMC Med Res Methodol.
2012;12:9.
48. Hayden  JA, van  der  Windt  DA, Cartwright  JL, Côté  P, Bombardier  C.
Assessing bias in studies of prognostic factors. Ann Intern Med.
2013;158(4):280–286.
49. Deeks  JJ, Higgins  JPT, Altman  DG. Analysing data and under-
taking meta-analyses. In: Higgins  JPT, Thomas  J, Chandler  J, et  al.,
eds. Cochrane Handbook for Systematic Reviews of Interventions.
Chichester: John Wiley & Sons; 2019:241–285.
50. Guyatt GH, Oxman AD, Vist GE, et al.; GRADE Working Group. GRADE:
an emerging consensus on rating quality of evidence and strength of re-
commendations. BMJ. 2008;336(7650):924–926.
51. Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG. Inactivation
of the DNA repair gene O6-methylguanine-DNA methyltransferase by
promoter hypermethylation is a common event in primary human neo-
plasia. Cancer Res. 1999;59(4):793–797.
52. Malley DS, Hamoudi RA, Kocialkowski S, Pearson DM, Collins VP,
Ichimura  K. A distinct region of the MGMT CpG island crit-
ical for transcriptional regulation is preferentially methyl-
ated in glioblastoma cells and xenografts. Acta Neuropathol.
2011;121(5):651–661.
53. Brigliadori G, Foca F, Dall’Agata M, et al. Defining the cutoff value of
MGMT gene promoter methylation and its predictive capacity in glio-
blastoma. J Neurooncol. 2016;128(2):333–339.