O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation
et al. (2011) O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome
in Malignant Glioma Independent of MGMT Promoter Methylation. PLoS ONE 6(2): e17156. doi:10.1371/journal.pone.0017156
6 O -Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation
Simone Kreth 0
Niklas Thon 0
Sabina Eigenbrod 0
Juergen Lutz 0
Carola Ledderose 0
Rupert Egensperger 0
Joerg C. Tonn 0
Hans A. Kretzschmar 0
Ludwig C. Hinske 0
Friedrich W. Kreth 0
Benjamin Rich, Dana-Farber Cancer Institute, United States of America
0 1 Department of Anaesthesiology, Ludwig Maximilians University , Munich, Germany , 2 Department of Neurosurgery, Ludwig Maximilians University , Munich, Germany , 3 Center for Neuropathology and Prion Research, Ludwig Maximilians University , Munich, Germany , 4 Department of Radiology, Ludwig Maximilians University , Munich, Germany , 5 Department of Anaesthesiology, University Medical Center Mannheim , Mannheim , Germany
Background: We analyzed prospectively whether MGMT (O6-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. Methodology/Principal Findings: Adult patients with a histologically proven malignant astrocytoma (glioblastoma: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p,0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p,0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p,0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p,0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression. Conclusions/Significance: MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined.
Funding: The work was funded by the departmental funds (Department of Anaesthesiology, Department of Neurosurgery, LMU Munich). The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
World Health Organisation (WHO) Grade III anaplastic
astrocytoma (AA) and WHO grade IV glioblastoma (GBM) are
rapidly progressive and resistant to therapy. Thus, malignant
glioma patients suffer the devastating effects of an incurable
disease with short survival times after diagnosis. More recently,
some progress has been achieved in the treatment of these tumors:
Prospective randomized studies of the European Organisation for
Research and Treatment of Cancer (EORTC) and the National
Cancer Institute of Canada (NCIC) trial have shown that the
addition of the alkylating agent temozolomide (TMZ) to
radiotherapy (XRT) for newly diagnosed GBM resulted in
significant prolongation of both time to progression and overall
survival. As a result, median survival which has been estimated in
the range one year for GBM and three years for AA [1,2,3] has
slightly been increased. Moreover, molecular markers have been
identified, which determine the course of the disease. An
important biomarker is the methylation status of the
O6methylguanine-DNA methyltransferase (MGMT) gene promoter.
Epigenetic silencing of the MGMT gene has been identified as a
strong and independent predictive factor of treatment response for
both GBM- and AA-patients undergoing chemotherapy with
alkylating agents [4,5]. Correlations between promoter
methylation and favorable treatment response after chemotherapy with
TMZ or other alkylating agents are explained by the assumption
that DNA methylation of a cysteine-phosphate-guanine (CpG)
island (CGI) within the MGMT promoter directly leads to a
repression of MGMT transcriptional activity and MGMT protein
expression ; determination of the promoter methylation status
may thus serve as a chemosensitivity sensor in glioma patients.
This hypothesis, however, which implies that MGMT promoter
methylation status, MGMT expression data and outcome
measurements are strongly correlated with each other, has not
unequivocally been supported: Studies evaluating MGMT
expression by immunohistochemistry (IHC), for example, mostly failed to
detect correlations between MGMT expression, MGMT
methylation status and outcome measurements . One more recently
published study on transcriptional activity in glioblastomas
questions mechanisms of direct transcriptional repression by
MGMT promoter methylation for a considerable number of
tumors: Even though overall a strong correlation between MGMT
promoter methylation and the degree of MGMT mRNA
expression was found , discordant findings were seen in at
least 15% of the investigated tumors, i.e. unmethylated
(methylated) tumors expressed low (high) levels of MGMT mRNA.
Unfortunately, this study did not provide any correlative data
between MGMT mRNA expression and clinical outcome to
further support the view of a sometimes broken link between
MGMT promoter methylation and mRNA expression.
The objective of the present study was to prospectively
investigate the predictive impact of MGMT gene expression under
consideration of its correlation with the MGMT promoter
methylation status in malignant glioma patients undergoing
XRT and/or TMZ treatment. As aberrant DNA
(cytosine-5)methyltransferase (DNMT) expression has been observed in
several tumor tissues  which might at least in part
explain epigenetic silencing of selected genes by promoter
methylation, we additionally estimated the expression of DNMTs
in tumor tissue as compared to normal brain, its prognostic/
predictive relevance in malignant glioma, and its correlation with
both the MGMT promoter methylation status and MGMT mRNA
Adult patients were eligible if they had i) a supratentorial GBM
or AA with histology being proven by stereotactic biopsy or open
tumor resection (May 2007 to March 2009), no prior history of
surgery, XRT, and/or chemotherapy, and a Karnofsky
performance score (KPS) $60 . All patients gave written informed
consent, and the prospective study protocol was reviewed and
approved by the institutional review board of the Ludwig
Maximilians University, Munich, Germany (AZ 216/14).
Indication for either surgical procedure was dependent on tumor size
and location, mass effects of the tumor, patients KPS and/or
significant co-morbidity. In case of moderate space occupying
effects of the tumor, a highly eloquent tumor location, and/or
significant co-morbidity stereotactic biopsy was preferred.
Histopathological diagnosis, determination of the MGMT promoter
methylation status and MGMT transcriptional activity were
obtained within 812 working days after surgery. Within 3 weeks
upon histopathological diagnosis, patients with GBM were
assigned to receive XRT plus concomitant and adjuvant TMZ
(XRT/TMZRTMZ). Treatment parameters were as follows:
XRT (60 Gy in 30 fractions)/TMZ (daily dose of 75 mg/
m2)RTMZ (150 to 200 mg/m2 per day for 5 days of every
28day cycle). In case of long term compliance, TMZ was continued
(at the same dose) until tumor progression occurred, which
indicated a difference to the EORTC treatment protocol .
Patients with the diagnosis of an AA were treated according the
EORTC protocol  in case of an extraaordinarily high Ki67
labelling index (.20%), otherwise primary chemotherapy with
TMZ was initiated and XRT was withheld . At baseline
evaluation, within 72 h after cytoreductive surgery, 46 weeks
after XRT/TMZ and every 3 cycles during TMZ maintenance
therapy, neuroradiologic examinations were performed. Early
treatment response was evaluated after the completion of 3 TMZ
cycles or earlier in case of clinical deterioration. Magnetic
resonance image (MRI) interpretation was independently done
according to the Macdonald criteria  by an experienced
neuroradiologist (JL), who was blinded for the MGMT methylation
status and transcriptional activity as well as for the follow up data
of the patients. Tumor progression had to be confirmed by further
clinical and neuroradiological follow up to exclude any bias by
pseudoprogression . Haematology was performed weekly.
Adverse events were defined according to the National Cancer
Institute (NCI) Common Toxicity Criteria, version 3.0. The
minimum follow up after inclusion of the last patient had to be 6
For histopathological evaluation, tissue samples harvested from
either cytoreductive surgery or biopsy procedures were fixed with
4% buffered formalin, paraffin embedded and subjected to routine
stainings (Hematoxylin and Eosin, Elastica van Gieson, Periodic
acid-Schiff) and IHC with antibodies against human GFAP
(monoclonal mouse, clone 6F2, Dako, Glostrup, Denmark) and
anti-MAP2 (clone HM-2, Sigma, Saint Louis, Missouri, USA).
Proliferation activity was determined using anti-human Ki67
antigen (mouse monoclonal, clone MIB-1, Dako, Glostrup,
Denmark). The histological diagnosis of all tissue specimens was
made according to WHO criteria .
Glioma tissue samples for molecular genetic analysis were
obtained from fluorescence-guided open tumor resections  or
serial stereotactic biopsy procedures [19,20]. Molecular genetic
evaluation of tissue samples obtained from open tumor resection
was exclusively done in tissue samples in the direct vicinity of
samples showing solid tumor tissue. In case of biopsy,
coregistration of computerized tomography (CT), and MRI
(including T1- and T2-weighted sequences) served for 3D
visualization (i-plan stereotaxyH, BrainLABH, Feldkirchen,
Germany) of the tumor and the simulation of the best biopsy trajectory
representative of the solid tumor. Serial biopsies were taken in
one-millimeter steps exactly along the chosen trajectory. Using
micro forceps the maximum amount of tissue per biopsy specimen
was 1 mm3. The number of specimens taken was in the range of
1018 samples per tumor. The tissue sampling procedure was
guided by intra-operative smear preparations, which were
routinely performed by the attending neuropathologist: Only
tumor probes next (i.e. 1 mm distance) to smear preparations
exclusively showing solid vital tumor tissue were selected for
molecular genetic analysis; a corresponding sample (level +1 mm),
which was taken for paraffin embedding and histopathological
examination using standard protocols , also had to show solid
vital tumor tissue. The described biopsy technique was chosen to
minimize the risk of tissue contamination (e.g. by non-neoplastic or
necrotic tissue) and more importantly, to recognize contamination,
if it occurs. For the detection of potential heterogeneity of MGMT
promoter methylation and MGMT mRNA expression throughout
the solid tumor space, biopsy specimens selected for
moleculargenetic analyses were harvested from at least two different sites
along the chosen trajectory of each tumor in the biopsy group.
Normal brain (from 9 patients) was obtained from epilepsy
surgery. One additional normal brain sample mRNA was
purchased from Ambion (Ambion, Austin, USA).
Combined RNA and DNA Isolation
A sequential purification procedure for both DNA and RNA
was performed as being published before . Briefly, RNA was
isolated using RNAqueousH Micro Kit (AmbionH, Austin, TX,
USA), and in a second step DNA was extracted using the QIAmpH
DNA Micro Kit (QiagenH, Hilden, Germany) from the first
flowthrough of RNA isolation following lysis of the sample. The
quantity and purity of the obtained nucleic acids was assessed
using the NanoDropH ND-1000 spectrophotometer (NanoDropH,
Wilmington, DE, USA).
Methylation-specific PCR (and sequencing analysis)
Exclusively histopathologically verified solid viable tumor tissue
was used for determination of MGMT promoter methylation and
measurements of transcriptional activity. Isolation of nucleic acids,
bisulfite modification of DNA, methylation-specific PCR (MSP)
and sequencing analyses were performed as being published in
detail before . In brief, DNA isolation from tumor specimens
was performed using commercially available isolation kits followed
by purification and bisulfite-modification of DNA . For MSP 2
pairs of primers, each specific for either the methylated or the
unmethylated MGMT promoter region, were used as described by
Esteller and collegues . Unmethylated versus methylated
tumors were defined as described by Grasbon-Frodl et al. .
Linear amplification and reverse transcription of RNA
2050 ng of purified RNA of all samples were amplified using
the TargetAmp-Kit (Epicentre, Madison, Wisconsin, USA)
according manufacturers recommendations in order to obtain
RNA amounts suitable for gene expression analyses . The
resulting amplification factors were between 500 and 2500.
Hereafter, equal amounts of the different samples of amplified
RNA (1000 ng) were transcribed into cDNA. The RT reaction
was carried out using random primers and Superscript III reverse
transcriptase (Invitrogen, Carlsbad, USA), as per manufacturers
Real-time qPCR was performed in triplicates with the Light
Cycler 480 instrument (Roche Diagnostics, Mannheim, Germany)
using Roches qPCR Mastermix and highly specific Universal
ProbeLibrary assays (Roche Diagnostics). The following primers
were used: MGMT: 59-
GTGATTTCTTACCAGCAATTAGCA39 (forward primer), 59- CTGCTGCAGACCACTCTGTG-39
(reverse primer); Probe: Universal ProbeLibrary probe: #52. TBP:
59- GAACATCATGGATCAGAACAACA-39 (forward primer),
59- ATAGGGATTCCGGGAGTCAT-39 (reverse primer); Probe:
Universal ProbeLibrary probe: # 87. SDHA: 59-
GAGGCAGGGTTTAATACAGCA-39 (forward primer), 59-
CCAGTTGTCCTCCTCCATGT-39 (reverse primer); Probe: Universal
ProbeLibrary probe: # 132. DNMT1: 59-
GATGTGGCGTCTGTGAGGT-39 (forward primer), 59-
CCTTGCAGGCTTTACATTTCC-39 (reverse primer); Probe: Universal ProbeLibrary probe:
# 66. DNMT3a: 59- ACTACATCAGCAAGCGCAAG -39
(forward primer), 59- CACAGCATTCATTCCTGCAA-39
(reverse primer); Probe: Universal ProbeLibrary probe: # 75.
DNMT3b: 59- CCGAGAACAAATGGCTTCAG-39 (forward
primer), 59- TTCCTGCCACAAGACAAACA-39 (reverse
primer); Probe: Universal ProbeLibrary probe: # 64. All assays were
designed intron-spanning. The thermal cycler conditions
comprised 45 cycles of 95uC for 10 s, 60uC for 30 s, and 72uC for 15 s.
Relative mRNA expression was calculated with the Relative
Quantification Software (Roche Diagnostics) using an
efficiencycorrected algorithm with standard curves and reference gene
normalization against SDHA and TBP (inclusion of a third
housekeeping gene (ACTB) led to similar results); These two
housekeeping genes have previously been shown to be appropriate
for normalization in human glioma and normal brain tissue .
The reference point of this study was the date of surgery.
Primary endpoint was progression free survival (PFS). Secondary
endpoints were overall survival (OS) and treatment response (TR).
We assumed the predictive impact of mRNA expression to be at
least as high as the impact of the MGMT promoter methylation
status. Values of MGMT mRNA expression in the biopsy group
usually referred to the mean of the expression data obtained from
different sites of each tumor. The median of the MGMT mRNA
expression of the entire tumor group was used as the cut-off value
for definition of the high and the low MGMT mRNA expression
group. Based on a previous study of our group  we expected a
hazard ratio of 0.45 or even less in favor of the group harboring a
methylated MGMT promoter and/or low MGMT mRNA
expression. Accordingly, a sample size in the range of 28 patients
in each group was estimated to be sufficient to have a power of
80% to demonstrate a significant difference in PFS in favor of
malignant glioma with a methylated MGMT promoter and/or low
PFS and OS were analyzed by the Kaplan-Meier method 
and compared with the two-sided log-rank test. TR was evaluated
after three cycles of TMZ monotherapy according to the
McDonald criteria . The Cox model was fitted to asses the
prognostic value of the MGMT methylation status, MGMT
mRNA expression, and other potential prognostic factors. First,
the importance of each variable was tested univariately. Forward
and backward step-wise proportional hazards modelling was
performed to assess the relative and independent prognostic
capacity of each parameter. In case of strong interrelationships
between covariates, several models were tested and compared with
each other (by computing the maximized likelihood). The
association between prognostic factors and TR was analyzed with
logistic regression models. The distribution of patient- and
tumorrelated variables between MGMT promoter methylated and
unmethylated subgroups was analyzed by the chi-squared statistics
(for dichotomized variables) and the Wilcoxon test (for
continuously scaled variables). In the biopsy group, pair wise comparison
of MGMT mRNA data at distant tumor sites was done with the
paired T-test. P#0.05 was considered significant. All calculations
were performed using the SAS software package (version 9.2)
Validation of dependency between MGMT mRNA expression
and MGMT promoter methylation status was performed using
data from The Cancer Genome Atlas (TCGA) database (http:tcga.
cancer.gov). TCGA glioblastoma samples were supplied by the
Broad Institute at the Massachusetts Institute of Technology and
the USC Epigenome Center, University of Southern California,
USA using the Affymetrix HG-U133A microarray and Illumina
Infinium Human DNA Methylation 27 beach chip technology. A
total of 209 GBM-samples containing both methylation and gene
expression data for the MGMT gene was extracted. As level 3 data
was used, no additional statistical preprocessing was necessary.
The data encompass a total of 20 methylation sites within the
MGMT-gene. For consistency with our molecular-genetic analyses,
only methylation sites were considered that correlated best with
gene expression as described by Everhard et al.  and map to
the genomic region covered by the MSP-primers. Beta-values of
the remaining methylation sites were averaged for each sample.
The median of the beta-values was chosen as the cut-off to classify
a sample as being either methylated or non-methylated.
A total of 63 patients (33 men, 30 women) with a median age of
59 years (range, 2580 years) were included (Table 1). The median
KPS was 70 (range, 6090). Nineteen patients had deep-seated
tumor locations and 30 patients harvested left-sided tumors.
Thirty-seven patients underwent molecular stereotactic biopsy
procedures (including all patients with AA). Complete tumor
Treatment response* tumor control
low expression (#0.45)
high expression (.0.45)
resection as determined by early postoperative MRI was
achieved in 13/26 patients treated with open tumor resection.
Histological evaluation revealed a GBM in 53 patients and an AA
in 10 patients. All patients were assessable for both determination
of the MGMT methylation status and MGMT mRNA expression
analyses. Treatment included a median number of 6 TMZ cycles
for the whole study population, which caused grade 1/2 toxicity in
17 patients and grade 3 toxicity in 2 patients.
MGMT promoter methylation and MGMT mRNA
From one single biopsy specimen around 150800 ng of RNA
(260:280 ratio between 1.8 and 2.1) and 1.52 mg DNA were
harvested, mainly depending on the size of the individual biopsy
specimen. The overall frequency of MGMT promoter methylation
was 45% (32/63 patients). 8/10 patients with AA and 24/53
patients with a GBM exhibited a methylated MGMT promoter
(Table 1). The overall median of the MGMT mRNA distribution
was 0.45 (range: 0.041.2). In thirty-three tumors of the biopsy
group at least two samples per tumor (collected from distant sites)
were available for both determination of the MGMT promoter
methylation status and expression analyses (overall number of
tissue specimens: 72); the mean distance between the chosen
biopsy sites was 9 mm (range 338 mm). In the remaining four
tumors only one tissue sample was used for molecular-genetic
analysis, as the corresponding second ones were suspected to be
contaminated by necrotic tissue/blood and/or non-neoplastic
tissue (as assumed by the results of both the intraoperative and
paraffin embedded analyses of specimens in the direct vicinity of
these tissue samples). The MGMT promoter methylation profile
was homogeneous throughout the viable solid tumor space of
those 33 tumors investigated. MSP and bisulfite sequencing
exhibited always concordant results. Pairwise comparison of
MGMT mRNA expression at different intra-tumoral positions
revealed no significant differences (p = 0.79, data not shown).
The median of the low expression group (less equal 0.45) was
0.25, whereas it was 0.8 in the high expression group (.0.45) of
the whole study population. Normal brain exhibited the highest
expression levels of MGMT mRNA (median: 1.1, p,0.001,
GBM subpopulations that underwent either cytoreductive
surgery or stereotactic biopsy did not differ in terms of age,
KPS, MGMT promoter methylation status, levels of MGMT
mRNA expression, applied chemotherapy cycles, and the follow
up period. Left sided and/or multifocal tumors were significantly
more frequently seen in patients undergoing biopsy only (p,0.01).
Patients with AA were significantly younger (median, 55 versus 62
years; p,0.05), showed more frequently a methylated MGMT
promoter and low expression levels of MGMT mRNA. GBM
subpopulations with either a methylated or unmethylated MGMT
promoter and/or either low or high expression levels of MGMT
mRNA did not differ with regard to patients characteristics. The
frequency of MGMT promoter methylation and MGMT mRNA
expression levels was nearly identical in patients undergoing
biopsy only and open tumor resection (data not shown). The
degree of MGMT mRNA expression strongly correlated with the
MGMT promoter methylation status (p,0.0001): The median of
the mRNA expression distribution in methylated and
unmethylated tumors was 0.26 (range: 0.040.78) and 0.8 (range: 0.351.2),
respectively (Figure 1). Discordant findings were seen in 12 (19%)
patients: MGMT promoter methylation was associated with high
mRNA expression (.0.45) levels in 6 patients (median: 0.58, range
0.460.78), whereas low expression levels were seen (#0.45) in
another 6 patients harboring an unmethylated MGMT promoter
(median: 0.39 range 0.150.45). Noteworthy, discordant findings
only concerned patients with GBM.
Nearly identical results were obtained by analyzing a publicly
available dataset of the TCGA database; 104 out of 209 GBM
tissue samples were methylated. The overall median of the
MGMT mRNA expression was 5.57 and was congruously used to
classify a high and a low expression group. Consistent with our
findings, MGMT gene expression was strongly associated with
methylation status with a median of 4.92 (range 3.797.38) for
methylated and 6.19 (range 4.777.78) for unmethylated samples
(p,0.001, Figure 1b). Discordant findings were observed in 46 of
209 samples (22%), which is in accordance to our data (19%).
Differences in the range of expression values result from the two
different technologies and normalization techniques used
(realtime PCR vs. array data).
DNMTs mRNA expression
The mRNA levels of DNMT1, DNMT3a and DNMT3b were
analysed in 63 malignant glioma and 10 normal brain samples.
Both in normal brain tissue and in tumors DNMT1 was found to
be the most expressed methyltransferase (more than 10-fold more
expressed than DNMT3a and DNMT3b, Figure 2A). In tumor
tissue as compared to normal brain, DNMT1 and DNMT3b were
significantly upregulated (DNMT1: 2.5-fold, DNMT3b: 3.2-fold,
p,0.001, Figure 2B); the degree of upregulation did not correlate
with MGMT promoter methylation status and MGMT mRNA
expression. For DNMT3a only a trend towards upregulation was
detected (1.6-fold, p,0.05); however, the degree of upregulation
was more pronounced when stratifying tumors by MGMT
methylation status: Unmethylated tumors exhibited significant
higher DNMT3a mRNA levels than methylated tumors
(p = 0.003), and in unmethylated tumors, DNMT3a expression
was 2.3-fold increased (p,0.001) as compared to normal brain
(Figure 2B). The subgroup analysis of patients with GBM revealed
identical results (data not shown).
The median follow up time was 10.5 months for the survivors.
Forty out of 63 patients exhibited tumor progression and 24
patients died. Death was tumor-related in all patients. No patient
was lost to follow up. Fifty-seven patients underwent XRT/
TMZRTMZ treatment and 6 patients with AA primary TMZ
chemotherapy, respectively. Kaplan-Meier estimates for PFS and
OS of the whole study population are presented in Figure 3A.
Treatment response (partial remission or stable disease) was seen
in 38/63 patients. Clinical outcome was in favor of MGMT
promoter methylated tumors and low MGMT mRNA expression:
Overall, early treatment response was significantly associated with
low MGMT expression (p = 0.004), whereas the influence of
MGMT promoter methylation was less pronounced (p = 0.02) and
even lost for the subgroup of patients with GBM; In the GBM
subgroup 19/24 patients with low expression and 12/29 patients
with high expression exhibited tumor control or tumor shrinkage
three months after XRT/TMZ (p,0.01). Overall, treatment
responders experienced a longer OS than non-responders (one
year survival rate: 86% vs. 30%, p,0.0001). Promoter
methylation correlated with both superior median PFS (18.3 versus 4.9
months) and OS (.22 versus 9.6 months; p,0.0001; Figure 3B).
Among patients with a methylated MGMT promoter the
unadjusted hazard ratio for disease progression and death was
0.22 (95% confidence interval: 0.110.46) and 0.2 (95%
confidence interval: 0.10.47). Stratification for low (#0.45) vs.
high (.0.45) MGMT mRNA expression levels also resulted in a
strong correlation with median PFS (17.5 vs. 5 months) and OS
(.20 vs. 9.5 months, p,0.0001, Figures 4A, 4B); The unadjusted
hazard ratio for disease progression and death was 0.32 (95%
confidence interval: 0.140.5) and 0.15 (95% confidence interval:
0.060.35). Exclusion of anaplastic tumors resulted in nearly
identical results concerning the prognostic/predictive impact of
both MGMT promoter methylation and MGMT mRNA
expression (data not shown). In the subgroup of GBM patients with
discordant findings stratification for mRNA expression resulted in
significant differences for both PFS and OS in case of a methylated
MGMT promoter: Methylated tumors with high mRNA
expression (N = 6) resulted in both shorter PFS (p,0.001, Figure 4C) and
OS (p,0.001, Figure 4D) than those with low mRNA expression
Figure 3. Kaplan-Meier estimates of 63 patients with malignant glioma. Tumor tissue obtained either by stereotactic biopsy or by open
surgery. A: Progression free survival and overall survival of the whole study population, B: Overall survival stratified by the MGMT promoter
Figure 4. Kaplan-Meier estimates of patients with malignant glioma stratified by MGMT mRNA expression. Tumor tissue obtained
either by stereotactic biopsy or by open surgery A: Progression free survival (N = 63), B: Overall survival (N = 63), C: Progression free survival of
patients with methylated GBMs (N = 24) D: Survival of patients with methylated GBMs (N = 24).
(N = 18): median PFS and OS was 17.5 months and 21.6 month
for the low-expression group, whereas it was 3.3 months and 10.4
months for the high expression group; PFS and OS were similar to
that of unmethylated tumors with high MGMT mRNA expression
(p.0.3). Conversely, unmethylated GBMs with low mRNA
expression (N = 6) did better than those with high mRNA
expression (N = 21, data not shown) in term of PFS, and OS;
the differences, however, were statistically not significant
(p = 0.06); Both PFS and OS was not significantly different to
that of methylated tumors with low mRNA expression (p.0.15).
Univariately, MGMT promoter methylation (p = 0.0001), low
mRNA (p = 0.0004) expression, AA histology (p,0.05) were
positively correlated with both increased PFS and OS. Open
tumor resection gained prognostic relevance in the subgroup of
patients with GBM (p = 0.03). No association was seen between
mRNA expression of DNMTs and clinical outcome. Multivariate
models including mRNA expression reached a fit as good as those
including MGMT promoter methylation; it allowed, however, the
inclusion of additional variables such as histology and type of
surgery (Table 2). The adjusted hazard ratios of MGMT promoter
methylation and mRNA expression for PFS and OS were
consistent with the unadjusted hazard ratios.
Daily clinical practise sometimes indicates discordance between
expectations derived from MGMT promoter methylation and
outcome, and one more recently published study on
transcriptional activity in glioblastomas has questioned mechanisms of
direct transcriptional repression by MGMT promoter
methylation for a considerable number of tumors: Unmethylated
(methylated) tumors were found to express low (high) levels of
MGMT mRNA in 15% of the study population . The results
of the current study are in line with the findings described by
Everhard et al. and an additionally performed analysis of an
independent validation dataset extracted from the TCGA
database. Furthermore, we demonstrate that MGMT mRNA is
homogeneously expressed throughout the solid tumor of
malignant gliomas, can be reliably determined even from small sized
biopsy specimens, is strongly correlated with outcome
measurements (even for those with discordant findings), and plays a direct
role for mediating tumor sensitivity to alkylating agents. Overall,
Progression Free Survival (PFS)
Low MGMT mRNA expression
Low MGMT mRNA expression
patients with low MGMT mRNA expression scores did
significantly better in terms of TR, PFS, and OS than those with high
expression scores. In particular, MGMT mRNA expression
retained influence even in those with discordant findings (19%
of the series): 6 patients harbouring methylated tumors with high
MGMT mRNA expression scores did significantly worse in terms
of PFS and OS than their 26 counterparts with concordant
findings; outcome was similar to that of unmethylated tumors with
high MGMT mRNA expression. A similar pattern was seen in 6
patients with an unmethylated MGMT promoter and low MGMT
mRNA expression. More data are needed to support the
hypothesis that in case of discordant findings expression data
could powerfully predict outcome independent of the MGMT
promoter methylation status.
Concerning the mechanisms underlying the discordant findings,
it may be hypothesized that a high MGMT mRNA expression
despite a methylated promoter might be due to overruling factors
such as increased NF-kB activity ; low MGMT expression
levels combined with an unmethylated promoter might result from
transcript destabilization and/or transcription-repressing factors,
such as miRNA regulation or histone modifications. However,
these issues need to be investigated and in this context, the here
described evaluation of MGMT transcriptional activity might be a
useful tool. The significant higher MGMT mRNA expression in
normal brain (which exhibits an unmethylated MGMT promoter)
as compared to that of unmethylated tumors also indicates the
existence of further mechanisms regulating MGMT expression
beyond promoter methylation.
As MSP and bisulfite sequencing do not cover all possible CpG
sites of the MGMT promoter, it cannot be excluded that omissions
of functionally relevant CpG sites may have partly accounted for
the detected discrepancies . However, even though some CpG
regions have been shown to reflect somewhat better MGMT
expression (range of concordant results: 7285%) in one more
recent study , no statistically significant difference could be
detected for any of the CpG regions investigated: All CpG sites
(including those studied by MSP) were highly correlated with
MGMT mRNA expression.
As aberrant DNMT expression has been observed in several
tumor tissues which might at least in part explain epigenetic
silencing of selected genes, we estimated the expression of DNMTs
in tumor tissue as compared to normal brain, its prognostic/
predictive relevance, and its correlation with both the MGMT
promoter methylation status and MGMT mRNA expression data.
In mammals, CGI methylation processes are regulated by
DNMT1 (maintenance of DNA methylation pattern) and
DNMT3a and DNMT3b (de novo methylation) . Aberrant
DNMT expression has been observed in various tumor entities
relative to normal tissue samples, indicating deregulation of
methylation processes in these tumors. For some tumor entities,
such as lung carcinoma [29,30], a correlation between DNMT
expression and clinical course was shown. Data describing DNMT
mRNA expression in malignant glioma are extremely scarce,
indicating an up-regulation of at least DNMT1 and DNMT3b in
GBM tissue samples as compared to normal brain . Neither
the prognostic/predictive impact of DNMT expression in
malignant glioma nor its association with MGMT promoter
methylation has been analyzed so far. In the current study, we
show that in malignant glioma DNMT1 and DNMT3b were
significantly upregulated, as compared to normal brain. The
degree of upregulation, however, did neither correlate with
outcome measurements, nor with MGMT promoter methylation
status or MGMT mRNA expression. For DNMT3a, only a slight
upregulation was detected. Interestingly, unmethylated tumors
exhibited significantly higher DNMT3a mRNA levels than
methylated tumors. Hence, regulation of the MGMT CGI
methylation by DNMT3a appears unlikely.
Taken together, the significant upregulation of DNMT1 and
DNMT3b indicates their involvement in CGI methylation
processes in malignant glioma. However, lack of correlation with
clinical outcome makes it reasonable to assume that yet unknown
additional mechanisms contribute to the degree of MGMT
promoter methylation. This aspect certainly deserves further
We previously showed that MSP and sequence analysis of
bisulfite-modified DNA for the determination of the MGMT
promoter methylation status revealed identical and reproducible
results throughout the solid tumor space, even for small amounts
of starting DNA as are obtained from a single 1-mm3 stereotactic
biopsy sample of a malignant glioma [20,24]. In the current series
a previously described new method of combined isolation
technique  of both RNA and DNA from a single 1-mm3
stereotatcatic biopsy sample was used for the first time for MSP,
sequence analysis and qPCR. As the extraction of high-quality
RNA is the limiting factor in the combined isolation of DNA and
RNA, a protocol was used that starts with RNA purification
followed by DNA recovery. DNA recovery was approximately
30% reduced compared with routine extraction techniques (i.e.
0.51 mg vs 11.5 mg from a 1-mm3 sample) suggesting that there
is a significant DNA loss in the RNA isolation procedure. This loss
of DNA, however, appears less relevant for tumors with increased
cellularity (such as malignant gliomas). In the current series tissue
specimens were snap frozen or processed directly upon withdrawal
to guarantee high quality of RNA. qPCR experiments were
performed according to the newest MIQE (Minimum Information
for Publication of Quantitative Real-Time PCR Experiments)
guidelines : All qPCR reactions were efficiency corrected and
data were normalized to the geometric mean of two reference
genes being determined as suitable for gene expression analyses in
human glioma and in glioma compared to normal brain tissue in
one of our previous studies . The similar rate of MGMT
promoter methylation and the similar degree of MGMT mRNA
and DNMTs expression in tissue samples obtained from both open
tumor resection and molecular stereotactic biopsy technique, and,
additionally, the reproducibility of these findings throughout the
solid tumor space underscored the validity of the applied methods.
It was shown that the applied biopsy technique allows avoiding the
contamination of tumor tissue by non-neoplastic tissue in the vast
majority of tumors of this series and, more importantly, to
recognize contamination if it occurs. It has been reported that
lymphocytes, endothelial cells and other types of intra-tumoral
non-neoplastic cells such as macrophages/microglias harbouring
all an unmethylated MGMT promoter might easily bias the
determination of both the MGMT promoter methylation status
and MGMT mRNA expression analysis . The reproducibility
of our results throughout the solid tumor space, however, indicates
that the impact of these intra-tumoral non-neoplastic cells must be
considered minor as compared to that of the solid viable tumor
tissue component. However, given the high expression of MGMT
mRNA in normal brain the necessity for collecting tissue samples
in a highly controlled fashion is underscored . Results
concerning the role of MGMT protein expression have been
shown to be not conclusive with regard to its correlation with
MGMT promoter methylation, MGMT mRNA expression, and
outcome measurements; inter-observer variability of IHC
evaluation, and varying specificity and sensitivity of antibodies might
contribute to the observed discrepancies .
Taken together our results show, in accordance to current
clinical experience, that MGMT promoter methylation status alone
does not suffice to provide information about the anticipated
clinical course in malignant glioma patients undergoing
chemotherapy with alkylating agents. Discordant findings between
MGMT promoter methylation status and MGMT mRNA
expression underscore the necessity to elucidate
methylationindependent mechanisms that may regulate MGMT expression.
We thank J. Rink and G. Groeger for their valuable technical assistance.
We would like to thank the TCGA team and associate research facilities for
the kind permission to use part of the glioblastoma data.
Conceived and designed the experiments: SK FWK. Performed the
experiments: SK SE CL RE. Analyzed the data: SK FWK. Contributed
reagents/materials/analysis tools: JCT JL NT HK. Wrote the manuscript:
SK FWK NT. Setting up and performing bioinformatical analyses: LCH.
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