ArticlePDF Available

Local delivery of temozolomide via a biologically inert carrier (Temodex) prolongs survival of glioma patients irrespectively of the MGMT methylation status.


Glioma is the most common brain malignancy. Standard first-line therapy for glioma includes surgery, radiotherapy and systemic administration of temozolomide. However, temozolomide does not reach the brain in sufficient doses when administered orally and has poor efficiency in more than half of the patients. Strategies to improve the treatment of glial malignancies are therefore needed. We have recently developed a system (Temodex) for local administration of temozolomide by encapsulating the drug in a biologically inert matrix. Here, we assessed the effect of Temodex in combination with standard therapy in a small-scale clinical study. Since the efficacy of temozolomide therapy is known to depend on the methylation status of the O6-methylguanine-DNA methyltransferase gene (MGMT) promoter, we also analyzed whether the effect of Temodex was influenced by the methylation status of MGMT. Our data show that the combination of standard therapy and Temodex was more efficient than standard therapy alone, promoting the overall patient survival by up to 33 weeks. Moreover, the efficacy of Temodex was not dependent on the methylation status of MGMT. Local Temodex administration in combination with standard therapy thereby emerges as a novel therapeutic option, with applicability that is independent on the methylation status of the MGMT promoter.
Running title: Local temozolomide in glioma patients 4
Local delivery of temozolomide via a biologically inert carrier (Temodex) prolongs survival of 6
glioma patients irrespectively of the MGMT methylation status. 7
1Double Bond Pharmaceutical AB, Uppsala, Sweden; 2Health Care Facility, Minsk City Emergency 12
Clinical Hospital, Minsk, Belarus; 3Department of Neurology and Neurosurgery, Belarusia n State 13
Medical University, Minsk, Belarus; 4Research Institute for Physical Chemical Problems, Belarusian 14
State University, Minsk, Belarus; 5Department of Medical Biochemistry and Microbiology, Uppsala 15
University, Uppsala, Sweden; 6Department of Anatomy, Physiology and Biochemistry, Swedish 16
University of Agricultural Sciences, Uppsala, Sweden 17
*Corresponde nce: 19
Received June 13, 2018 / Accepted August 28, 2018 21
Glioma is the most common brain malignancy. Standard first-line therapy for glioma includes surgery, 24
radiotherapy and systemic administration of temozolomide. However, temozolo mide does not reach the 25
brain in sufficient doses when administered orally and has poor efficiency in more than half of the 26
patients. Strategies to improve the treatment of glial malignancies are therefore needed. We have 27
recently developed a system (Temodex) for local administratio n of temozolomide by encapsulating the 28
drug in a biologically inert matrix. Here, we assessed the effect of Temodex in combination with 29
standard therapy in a small-scale clinical study. Since the efficacy of temozolomide therapy is known 30
to depend on the methylation status of the O6-methylguanine-DNA methyltransferase gene (MGMT) 31
promoter, we also analysed whether the effect of Temodex was influenced by the methylation status of 32
MGMT. Our data show that the combination of standard therapy and Temodex was more efficient than 33
standard therapy alone, promoting the overall patient survival by up to 33 weeks. Moreover, the 34
efficacy of Temodex was not dependent on the methylation status of MGMT. Local Temodex 35
administratio n in combination with standard therapy thereby emerges as a novel therapeutic option, 36
with applicability that is independent on the methylation status of the MGMT promoter. 37
Key words: glioma, Temodex, temozolomide, MGTM, methylation 39
Gliomas constitute the most common fo rm of brain tumours in adults. Despite the considerable efforts 41
that have bee n invested into the deve lopment of improved therapeutic regimens for glioma, these 42
malignancies are continuing to b e associated with a notoriously poor prognosis [1]. Therefore, 43
regimens to prolong survival in this disease are urgently needed. The standard therapy for glioma 44
(Stupp therapy) includes surgery, radiotherapy and systemic administration of temozolomide [2]. 45
Although the penetration of temo zolomide into brain from plasma is better compared to other drugs, it 46
is still limited, with the ratio of maximum plasma over brain tumour concentration of temozolomide 47
after an oral administration being below 20% [3]. Moreover, to achieve therapeutic levels in brain, 48
temozolomide must be administered in high systemic doses due to its short half-life (~1.8h in plasma 49
[4]), which in turn requires prolonged systemic administration and leads to side effects such as 50
thrombocytopenia, nausea and vomiting [5]. 51
In order to achieve improved disease outcome, we have recently developed a system for sustained local 52
delivery of temozolomide, by encapsulating the drug in a biologically degradable matrix. This system 53
of temozolomide delivery (Temodex) has recently been approved for clinical use in Belarus (reg. 54
number 14/12/2324, 55
Previous studies have suggested that the outcome of temozo lomide therapy is depend ent on the 56
methylation status of the O6-methylguanine-DNA methyltransferase gene (MGMT) [6, 7]. MGMT is an 57
enzyme that is crucial for repairing DNA dama ge, including damage caused by genotoxic e ffects of 58
chemotherapeutic drugs such as temozolomide. Hence, patients with active transcription o f MGMT in 59
their tumour tissue are known to be relatively resistant to effects of t emozolomide whereas patients 60
where MGMT transcription has been silenced by hypermethylation of the MGMT promoter are 61
sensitive [8-10]. 62
In this study, the aim was to evaluate the impact of Temodex on the clinical outcome of glioma, and to 63
determine if the effect of Temodex is dependent on the methylation status of the MGMT promoter. For 64
this purpose, we stud ied the effects o f Temodex in a limited co hort of patients whose MGMT 65
methylation status and expression of the MGMT protein had been characterized. Our results indicate a 66
positive effect of Temode x and that its positive effect on overall survival may be independent of the 67
methylation status of the MGMT promoter. 68
Patients and methods 70
Study se tup. Paraffin-embedded tumour tissue samples were collected from patients with brain 71
tumours d uring clinical trials o f Temodex at the Emergency Hospital #5 in Minsk, Belarus. The study 72
was performed according to the ethical approva l in protocol #14 from 2012.07.30 issued by the ethical 73
committee of the hospital, and the study protocol including the patient informed consent and treatment 74
of data and samples, were performed according to the Declaration of Helsinki. The samples were 75
assessed for MGMT promoter methylation status and MGMT protein content. The analyses were 76
blinded and were performed at Rigshospitalet (Copenhagen, Denmark). In tota l, 100 samples from 78 77
patients were analysed, of which 29 patients received both standard care Stupp therapy (surgery, 78
radiotherapy and systemic temozolomide (100 mg/patient) a nd Temodex (Temodex group; 900 79
mg/patient), whereas 49 patients received standard of care Stupp therapy only (Control group). 80
Temodex is a formulatio n of temo zolomide encapsulated in a biodegradable matrix. Upon contact with 81
an aq ueous milieu, Temodex forms a viscous gel that retains temozo lomide at the site of 82
administration, allowing for a slow release and e nsuring that the edges of the wound are continuo usly 83
exposed to temozolomide. The samples from the Temodex patient group were collected between 84
November 2012 and December 2013, and samples from the control group were taken between October 85
2009 and October 2012. Patients were followed for up for 36 to 60 mo nths and the o verall s urvival of 86
the cohort was assessed. Temodex is approved for clinical use in Belarus since October 2014. 87
Sample prepa ration. Paraffin blocks were first re-paraffinized into new blocks and the n sectioned for 88
ining and MGMT protein staining. The adjacent 89
30- MGMT gene promoter methylation analyses. 90
MGMT promoter methylation ass essment. Sodium b isulfite conversion o f the samples was 91
performed using the Epitect bisulfite k it (Qiagen, Hilden, Germany) according to the manufac tures' 92
instructions. PCR and p yrosequencing were performed using the Therascreen MGMT Pyro kit 93
(Qiagen) according to the manufactures' instructions. For each pyrosequencing run, three controls were 94
included: One non-template co ntrol (NTC) from PCR, one methylated DNA control provided with the 95
kit and one sample o f DNA from an anonymous healthy blood donor (nonmethylated DNA). For 96
assigning patients into groups of hypermethylated or nonmethyla ted MGMT promoter status two 97
different thresho lds were used: a mean methylation leve l of 8% [7] or 10% [11], i.e. methylatio n levels 98
above either 8 or 10% were considered as hypermethylation. 99
Immunohistochemistry. For immunohistoche mistry analyses, formalin-fixed paraffin embedded 100
sections were deparaffin ized in xylene and rehydrated in decreasing concentrations of ethanol. After 101
blocking of endogenous peroxidase with 3 % H2O2, the sections were pre-treated in a microwave o ven 102
with a Tris-EGTA-buffer, and immunostained on a DAKO Cytomation autostainer using a monoclonal 103
mouse anti-MGMT antibody (MAB16200, 1:200, Millipore, USA). Immunoreactivity was visualized 104
with DAB+ (DAKO K3468) as chromoge n. The immunohis tochemical stainings were semi-105
quantitatively eva luated according to the number of tumour cells stained: 0-25% = negative 106
(methylated MGMT), and >26% = positive (nonmethylated MGMT). For MGMT evaluation, positive 107
endothelia l cells, lymphocytes, and microglia served as positive internal control. 108
Statistical a nalysis. Baseline data were divided by MGMT status (gene methyla tion and 109
immunohistochemistry) and assessed using Kaplan-Meier survival analyses (log-rank test) of the 110
Temodex group compared to the control group, and o f the patients that were MGMT hypermethylation-111
positive according to both DNA methylation grade and the IHC staining of the MGMT protein in the 112
tumor tissue sections. For these analyses, Graph Pad Prism 4.0c software was used. Kap lan-Meier plots 113
were used to present the outcome of the analyses. Any differences were conside red statistica lly 114
significant when the p-value was < 0.05. 115
Results 117
The clinical data for the p atients evaluated in this study are s hown in Table 1. Notably, out of a total of 118
78 patients, samples from 13 patients from the Temodex group and 26 from the control group were 119
assessable for both MGMT methylation a nd tumor tissue protein e xpression (Supplementary Table 1). 120
Represe ntative staining for MGMT protein are shown in Figure 1. Data from the patients in which 121
MGMT protein was assessed and where also the MGMT promoter methylation status could be 122
determined were further evaluated. In the Temodex group, DNA from eight of the samples was pos itive 123
for MGMT promoter methylation, whereas DNA from five samples was no nmethylated. In the control 124
group, DNA from 19 of the samples was positive for MGMT promoter methylation whereas seve n were 125
nonmethylated. 126
Patients were treated with either standard therapy (surgery, radiotherapy, systemic temo zolomide) or 127
with a combination of standard therapy and a single dose of locally administered temozolomide 128
(Temodex). The patients involved in these analyses had all been classified according to MGMT 129
methylation status, as judged b y both DNA methylatio n levels (see also below) and as judged by 130
MGMT protein positivity. MGMT protein positivity is a sign of nonmethylated MGMT promoter 131
whereas MGMT negativity is regarded as a sign of hypermethylated MGMT. The reason for evalua ting 132
both of these parameters is that previous studies have indicated that both of these parameters are of 133
importance for determining outcome of treatment [8, 12, 13]. MGMT protein positivity was assessed 134
by immunohistochemical analyses (Figure 1). 135
Hype rmethylation status and survival. The DNA hypermethylation threshold of the MGMT 136
promoter was set at either 10% or 8%, based on previo us studies suggesting that either 10% [8] or 8% 137
[14-16] can be considered as relevant cut-offs to discriminate between non- and hypermethylated 138
MGMT. Only samples whe re the MGMT methylation status was supported by bo th DNA ana lysis and 139
by protein staining were included in the analyses. 140
We first analysed the effect of Temodex by including all of the patients, i.e. both those displaying 141
MGMT hypermethylation and those negative for MGMT hypermethylation (Figure 2). When applying 142
the analysis to patients with a 10% c ut-off for DNA methylatio n, the results reveal that patients treated 143
with Temodex in addition to standard of care treatment had 14.2 weeks longer overall survival 144
compared to patients that received standard of care treatment only (p=0.036; Figure 2A). Under these 145
conditions, the med ian overall survival was 41.36 weeks for the control group vs. 55.57 weeks for the 146
Temodex group. Also when applying 8% as the hypermethylatio n cut-off, a pronounced effect of 147
Temodex o n overall survival was seen, with the Temodex treatment increasing the mean overall 148
survival by 33 weeks (p = 0.027, Figure 2B). In this setting, the median overall survival was 43.0 149
weeks for the control group vs. 89.43 weeks for the Temodex group. Data from patients where only 150
MGMT methylation status or o nly MGMT protein positivity could be assessed (not both) were also 151
analysed. In contrast to the patients where both of these parameters could be assessed, the Temodex 152
treatment did not affect the outcome (data not shown). 153
Efficacy of Temodex treatment. To evaluate if the efficacy of Temodex treatment was dependent o n 154
the methylation status of MGMT, the effect o f Temodex was evaluated on patients that had been 155
discriminated into being positive or negative for MGMT hypermethylation. Patients displaying MGMT 156
hypermethylation as judged by DNA analysis (with either 8% or 10% cut-off threshold) were denoted 157
MGMThm+, and those where MGMT hypermethylation was inferred by protein staining were denoted 158
MGMTp-. Patients negative for MGMT hypermethylation were indic ated as MGMThm- and MGMTp-, 159
respectively. 160
Among patients treated with Temodex, there were no significant d ifferences in overall survival 161
between patie nts positive for MGMT promoter hypermethylation a162
10% methylation cut-off) and protein staining results (MGMThm +p-) compared to patients negative for 163
MGMT methylation (MGMThm-p+) (Figure 3A). When the threshold for hypermethylation of the 164
MGMT ilar results were obtained, i.e. Temodex had a positive effect on 165
overall surviva l of patients irrespectively of the MGMT promoter methylation status (Figure 3B). 166
Hence, Temodex has a positive effect on overall survival of patients regardless of the me thylation 167
status of MGMT and irrespectively if the cut-off for hypermethylation is set at 8% or 10%. 168
Data from patients where only the MGMT methylation status or MGTM protein positivity had been 169
considered were assessed for differences in survival between control groups (hypermethylated vs. non-170
methylated), Temodex groups (hypermethylated vs. non-methylated), non-methylated groups 171
(Temodex vs. control) and hypermethylated groups (control vs. Temodex). Neither of these ana lyses 172
revealed statistically significant differences betwee n the groups, either at 8% or 10% cut-off for MGMT 173
methylation status (data not shown). 174
Discussion 176
Glioma continues to constitute a major pathology of the brain, afflicting a large number of subjects 177
worldwide. Moreover, there are currently few effective therapeutic options available for this type of 178
malignancy. In this study of relatively limited power, we show that the application of Temodex in 179
combination with standard therapy has a profound and positive effect on the overall surviva l of glioma 180
patients, with an increased survival time of up to 33 weeks. Thereby, the establishment of Temodex has 181
expanded the repertoire of available therapeutic options in glioma, and we envision that Temodex may 182
gain widespread use in the treatment of this malignancy. Moreover, we foresee that Temodex also may 183
become useful in the treatment of other types of malignancies of the brain.184
Intriguingly, we demonstrate that Te modex shows effic acy that is not dependent on the methylation 185
status of the MGMT promoter, i.e. that Temodex is equally effective in patients with low or high 186
expression of MGMT. This was somewhat unexpected considering previous studies indicating that 187
temozolomide has low efficacy in patients with active expression of MGMT as result of a low extent or 188
absence of MGMT promo ter hypermethylation [17-19]. The underlying reason for the latter notion is 189
that high MGMT expression results in efficient DNA repair such that the genotoxic effects o f 190
temozolomide are minimized [6]. Although we cannot at present with certainty explain why Te modex 191
shows high efficacy even in those patients with high expression of MGMT, it should be emp hasized 192
that Temodex is directly applied at the site of the tumor lesion, and that the local concentration of 193
temozolomide thereby will be high- considerably higher than when only administrating temozolomide 194
systemically. We may thus propose that such high, local concentratio ns of temozolomide may impose 195
more potent and rapid cytotoxic effects on the tumor cells than if the drug is merely administered 196
systematically. This may lead to rapid induction o f apoptosis such that the tumor cells fail to produce 197
MGMT at levels that are sufficie nt to carry out efficient DNA repair. 198
It sho uld be noted that the present study was performed o n a relatively small patient cohort, and t hat 199
extended studies on larger patient materials need to be carried out to firmly establish Temodex as a 200
robust treatment option in glioma. Nevertheless, the present study establishes Temodex as a potential 201
novel therapeutic agent to be used in combination with standard therapy in treatment of glioma patients 202
showing both high and low MGMT expression. It is also notable that the Temodex represents a 203
relatively cheap treatment option, and we therefo re foresee that Temodex may become a preferred 204
choice regardless of economic considerations. 205
Acknowledgements 207
We are grateful to Emergenc y Hospital #5 (Minsk, Belarus) for providing samples for this study. 208
Disclosures 210
IK and IL are emplo yed by Double Bond Pha rmaceutical. GP receives consulting fees from Double 211
Bond Pharmaceutical. 212
References 213
Epidemiology of glial a nd no n-glial brain tumours in Europe. Eur J Cancer 2012; 48: 1532-215
1542. 216
grade glioma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann 218
Oncol 2014; 25 Suppl 3: iii93-101. 219
neuropharma cokinetics of temozolomide in patients with resectable brain tumors: potential 221
implications for the current approach to chemoradiation. Clin Cancer Res 2009; 15: 7092-7098. 222 223
metabolism, and excretio n of 14C-temozolomide following oral administration to patients with 225
advanced cancer. Clin Cancer Res 1999; 5: 309-317. 226
[5] LAQUINTANA V, TRAPANI A, DENORA N, WANG F, GALLO JM et al. New strategies to 227
deliver anticancer drugs to brain tumors. Expert Op in Drug Deliv 2009; 6: 1017-1032. 228 229
silencing and benefit from temozo lomide in gliob lastoma. N Engl J Med 2005; 352: 997-1003. 231 232
[7] MCDONALD KL, RAPKINS RW, OLIVIER J, ZHAO L, NOZUE K et al. The T genotype of 233
the MGMT C>T (rs16906252) enhancer single-nuc leotide polymorphism (SNP) is associated 234
with promoter methylation a nd longer survival in glioblastoma patients. Eur J Cancer 2013; 49: 235
360-368. 236
Assessment of Quantitative and Allelic MGMT Methylation Patterns as a Prognostic Marker in 238
Glioblastoma. J Neuropathol Exp Neurol 2016; 75: 246-255. 239
[9] TANG K, JIN Q, YAN W, ZHANG W, YOU G et al. Clinical correlation o f MGMT protein 240
expression and promoter methylation in Chinese glioblastoma patients. Med O ncol 2012; 29: 241
1292-1296. 242
Promoter Methylation Is a Strong Prognostic Biomarker for Benefit from Dose-Intensified 244
Temozo lomide Rechallenge in Progressive Glioblastoma: The DIRECTOR Trial. Clin Cancer 245
Res 2015; 21: 2057-2064. 246
[11] XIE H, TUBBS R, YANG B. Detection of MGMT promoter methylation in glioblastoma using 247
pyrosequencing. Int J Clin Exp Pathol 2015; 8: 636-642. 248
[12] THON N, KRETH S, KRETH FW. Personalized treatment strategies in glioblastoma: MGMT 249
promoter methyla tion status. Onco Targets Ther 2013; 6: 1363-1372. 250 251
[13] WELLER M. Assessing the MGMT status in glioblastoma: one step forward, two steps back? 252
Neuro Oncol 2013; 15: 253-254. 253
methylation and expression of MGMT and the DNA mismatch repair genes MLH1, MSH2, 255
MSH6 and PMS2 in paired primary and recurrent glioblastomas. Int J Cancer 2011; 129: 659-256
670. 257
Predictive impact of MGMT promo ter methylation in glioblastoma of the elderly. Int J Cancer 259
2012; 131: 1342-1350. 260
promoter methylation determined by HRM in comparison to MSP and pyrosequencing for 262
predicting high- grade glioma response. Clin Epigenetics 2016; 8: 49. 263 264
[17] BRELL M, TORTOSA A, VERGER E, GIL JM, VINOLAS N et al. Prognostic significance of 265
O6-methylguanine-DNA methyltransferase determined by promo ter hypermethylation and 266
immunohistochemical expression in anaplastic gliomas. C lin Cancer Res 2005; 11: 5167-5174. 267 268
[18] HEGI ME, DISERENS AC, GODARD S, DIETRICH PY, REGLI L et al. Clinical trial 269
substantiates the predictive value of O-6-methylguanine-DNA methyltransferase promoter 270
methylation in glioblastoma patients treated with temozolomide. Clin Cancer Res 2004; 10: 271
1871-1874. 272
[19] ZHANG K, WANG XQ, ZHOU B AND ZHANG L. The prognostic value of MGMT promoter 273
methylation in Glioblastoma multiforme: a meta-analysis. Fam Cancer 2013; 12: 449-458. 274 275
Figures Legends 277
Figure 1. Immunhistoche mical analysis for MGMT protein. Tissue sections from brain tumour samples 278
were stained with an antibody to MGMT or with Hematoxylin and Eosin (H&E). Representative image 279
of MGMT-negative (A) MGMT-positive samples (B), and H&E staining of representative MGTM-280
positive samples (C) are shown. 281
Figure 2. Kaplan-Meier survival plo t for overall surviva l for patients treated with Temodex and 283
standard of care (Temodex) compared to patients treated with standard o f care only (Control). In these 284
analyses, only patients where both the MGMT protein positivity and MGMT methylation s tatus could 285
be assessable, and where these parameters showed consistency, were included. The MGMT methylation 286
threshold was set at over 10% (A) or 8% (B). 287
Figure 3. Kaplan-Meier survival plot for overall surviva l fo r patients treated with Temodex. Survival 289
of patients positive for MGMT hypermethylation and negative for MGMT protein staining in the 290
tumour tissue (Temodex MGMThm+) did not differ significantly from the survival o f patients without 291
MGMT hypermethylation and with positive MGMT protein staining in the tumour tissue (Temode x 292
MGMTh m-). The thresholds for hypermethylation were set at either 10% (A) or 8% (B). 293
Table 1. Clinical and demographic characteristics. 296
Study group
Control group
ResearchGate has not been able to resolve any citations for this publication.
Full-text available
Background The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value. Results We analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan–Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP. Conclusions Compared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.
Full-text available
Purpose Rechallenge with temozolomide (TMZ) at first progression of glioblastoma after temozolomide chemoradiotherapy (TMZ/RT→TMZ) has been studied in retrospective and single-arm prospective studies, applying TMZ continuously or using 7/14 or 21/28 days schedules. The DIRECTOR trial sought to show superiority of the 7/14 regimen. Patients & Methods Patients with glioblastoma at first progression after TMZ/RT→TMZ and at least 2 maintenance TMZ cycles were randomized to Arm A (one week on (120 mg/m2 per day) / one week off) or Arm B (three weeks on (80 mg/m2 per day) / one week off). The primary end point was median time to treatment failure (TTF) defined as progression, premature TMZ discontinuation for toxicity, or death from any cause. O6-methylguanine DNA methyltransferase (MGMT) promoter methylation was prospectively assessed by methylation-specific PCR. Results Because of withdrawal of support, the trial was prematurely closed to accrual after 105 patients. There was a similar outcome in both arms for median TTF (A: 1.8 months [95% CI 1.8-3.2] versus B: 2.0 months [95% CI 1.8-3.5]) and overall survival (OS) (A: 9.8 months [95% CI 6.7-13.0] versus B: 10.6 months [95% CI 8.1-11.6]). Median TTF in patients with MGMT-methylated tumors was 3.2 months [95% CI 1.8-7.4] versus 1.8 months [95% CI 1.8-2] in MGMT-unmethylated glioblastoma. Progression-free survival rates at six months (PFS-6) were 39.7% with versus 6.9% without MGMT promoter methylation. Conclusions TMZ rechallenge is a treatment option for MGMT promoter-methylated recurrent glioblastoma. Alternative strategies need to be considered for patients with progressive glioblastoma without MGMT promoter methylation. Copyright © 2015, American Association for Cancer Research.
Full-text available
The identification of molecular genetic biomarkers considerably increased our current understanding of glioma genesis, prognostic evaluation, and treatment planning. In glioblastoma, the most malignant intrinsic brain tumor entity in adults, the promoter methylation status of the gene encoding for the repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) indicates increased efficacy of current standard of care, which is concomitant and adjuvant chemoradiotherapy with the alkylating agent temozolomide. In the elderly, MGMT promoter methylation status has recently been introduced to be a predictive biomarker that can be used for stratification of treatment regimes. This review gives a short summery of epidemiological, clinical, diagnostic, and treatment aspects of patients who are currently diagnosed with glioblastoma. The most important molecular genetic markers and epigenetic alterations in glioblastoma are summarized. Special focus is given to the physiological function of DNA methylation-in particular, of the MGMT gene promoter, its clinical relevance, technical aspects of status assessment, its correlation with MGMT mRNA and protein expressions, and its place within the management cascade of glioblastoma patients.
Recent clinical trials on patients with glioblastoma revealed that O6-Methylguanine-DNA methyltransferase (MGMT) methylation status significantly predicts patient's response to alkylating agents. In this study, we sought to develop and validate a quantitative MGMT methylation assay using pyrosequencing on glioblastoma. We quantified promoter methylation of MGMT using pyrosequencing on paraffin-embedded fine needle aspiration biopsy tissues from 43 glioblastoma. Using a 10% cutoff, MGMT methylation was identified in 37% cases of glioblastoma and 0% of the non-neoplastic epileptic tissue. Methylation of any individual CpG island in MGMT promoter ranged between 33% and 95%, with a mean of 65%. By a serial dilution of genomic DNA of a homogenously methylated cancer cell line with an unmethylated cell line, the analytical sensitivity is at 5% for pyrosequencing to detect MGMT methylation. The minimal amount of genomic DNA required is 100 ng (approximately 3,000 cells) in small fine needle biopsy specimens. Compared with methylation-specific PCR, pyrosequencing is comparably sensitive, relatively specific, and also provides quantitative information for each CpG methylation.
The prognostic significance of O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation on Glioblastoma multiforme (GBM) remains controversial. A meta-analysis of published studies investigating the effects of MGMT promoter methylation on both progression-free survival (PFS) and overall survival (OS) among GBM patients was performed. A total of 2,986 patients from 30 studies were included in the meta-analysis. In all, the frequency of MGMT promoter methylation was 44.27 %. Five studies undertook univariate analyses and nine undertook multivariate analyses of MGMT promoter methylation on PFS. The pooled hazard ratio (HR) estimate for PFS was 0.72 (95 % CI 0.55-0.95) by univariate analysis and 0.51 (95 % CI 0.38-0.69) by multivariate analysis. The effect of MGMT promoter methylation on OS was evaluated in 15 studies by univariate analysis and 14 studies by multivariate analysis. The combined HR was 0.67 (95 % CI 0.58-0.78) and 0.49 (95 % CI 0.38-0.64), respectively. For GBM patients treated with Alkylating agent, the meta-risk remained highly significant by both univariate (HR = 0.58; 95 % CI 0.42-0.79) and multivariate analysis (HR = 0.42; 95 % CI 0.29-0.60). This study showed that MGMT promoter methylation was associated with better PFS and OS in patients with GBM regardless of therapeutic intervention, and associated with longer OS in GBM patients treated with alkylating agents.
Clinical studies in patients with newly diagnosed glioblastoma treated with temozolomide have shown that the methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is both predictive and prognostic of outcome. Methylation of the promoter region of MGMT is the most clinically relevant measure of MGMT expression and its assessment has become integral in current and planned clinical trials in glioblastoma. Our study confirmed that MGMT methylation, assessed by pyrosequencing, is associated with a significant survival benefit in glioblastoma patients treated with temozolomide (either concurrently with radiotherapy or sequential treatment). More interestingly, our study demonstrated that a promoter variant, the c.-56C>T (rs16906252) single nucleotide polymorphism (SNP) located within a cis-acting enhancer element at the proximal end of MGMT, is associated with the presence of MGMT promoter methylation in de novo glioblastoma. Furthermore, we show that the overall survival of patients carrying both the SNP and MGMT methylation showed a strong survival benefit when compared to either molecular event on their own. Promoter reporter experiments in MGMT methylated glioblastoma cell lines showed the T allele conferred a ∼30% reduction in normalised MGMT promoter activity compared to the wild-type haplotype. This might account for the propensity of the T allele to undergo promoter methylation, and in turn, the improved survival observed in carriers of the T allele. An independent validation on larger cohorts is required to confirm the prognostic and predictive value of individuals carrying the T allele.
To the central nervous system (CNS) belong a heterogeneous group of glial and non glial rare cancers. The aim of the present study was to estimate the burden (incidence, prevalence, survival and proportion of cured) for the principal CNS cancers in Europe (EU27) and in European regions using population-based data from cancer registries participating in the RARECARE project. We analysed 44,947 rare CNS cancers diagnosed from 1995 to 2002 (with follow up at 31st December 2003): 86.0% astrocytic (24% low grade, 63% high grade and 13% glioma NOS), 6.4% oligodendroglial (74% low grade), 3.6% ependymal (85% low grade), 4.1% Embryonal tumours and 0.1% choroid plexus carcinoma. Incidence rates vary widely across European regions especially for astrocytic tumours ranging from 3/100,000 in Eastern Europe to 5/100,000 in United Kingdom and Ireland. Overall, about 27,700 new rare CNS cancers were estimated every year in EU27, for an annual incidence rate of 4.8 per 100,000 for astrocytic, 0.4 for oligodendroglial, 0.2 for ependymal and embryonal tumours and less than 0.1 for choroid plexus carcinoma. More than 154,000 persons with rare CNS were estimated alive (prevalent cases) in the EU at the beginning of 2008. Five-year relative survival was 14.5% for astrocytic tumours (42.6% for low grade, 4.9% for high grade and 17.5% for glioma NOS), 54.5% for oligodendroglial (64.9% high grade and 29.6% low grade), 74.2% for ependymal (80.4% low grade and 36.6% high grade), 62.8% for choroid plexus carcinomas and 56.8% for embryonal tumours. Survival rates for astrocytic tumours were relatively higher in Northern and Central Europe than in Eastern Europe and in UK and Ireland. The different availability of diagnostic imaging techniques and/or radiation therapy equipment across Europe may contribute to explain the reported survival differences. The estimated proportion of cured patients was 7.9% for the 'glial' group to which belong astrocytic tumours. Overall results are strongly influenced by astrocytic tumours that are the most common type. This is the first study to delineate the rare CNS cancer burden in Europe by age, sex and European region.
O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter methylation identifies a subpopulation of glioblastoma patients with more favorable prognosis and predicts a benefit from alkylating agent chemotherapy (CT). Little is known about its prevalence and clinical significance in older glioblastoma patients. We studied 233 glioblastoma patients aged 70 years or more (144 males, 89 females, median age: 74 years, range: 70.0-86.6 years), who were prospectively enrolled in the German Glioma Network, for MGMT promoter methylation by methylation-specific PCR (MSP) in all patients and DNA pyrosequencing in 166 patients. MGMT data were correlated with patient outcome. Median progression-free survival (PFS) was 4.8 months (95% CI: 4.3-5.3) and median overall survival (OS) was 7.7 months (95% CI: 6.3-9.0). MGMT promoter methylation was detected by MSP in 134 patients (57.5%). For the whole cohort, PFS was 5.2 versus 4.7 months (p = 0.207) and OS was 8.4 versus 6.4 months (p = 0.031) in patients with versus without MGMT promoter methylation. Patients with MGMT methylated tumors had longer PFS when treated with radiotherapy (RT) plus CT or CT alone compared to patients treated with RT alone. Patients with MGMT unmethylated tumors appeared to derive no survival benefit from CT, regardless of whether given at diagnosis together with RT or as a salvage treatment. Patients treated with RT plus CT or CT alone demonstrated longer OS when pyrosequencing revealed >25% MGMT methylated alleles. Taken together, MGMT promoter methylation may be a useful biomarker to stratify elderly glioblastoma patients for treatment with versus without alkylating agent CT.
Epigenetic silencing of the O(6) -methylguanine-DNA methyltransferase (MGMT) gene promoter is associated with prolonged survival in glioblastoma patients treated with temozolomide (TMZ). We investigated whether glioblastoma recurrence is associated with changes in the promoter methylation status and the expression of MGMT and the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 in pairs of primary and recurrent glioblastomas of 80 patients, including 64 patients treated with radiotherapy and TMZ after the first operation. Among the primary tumors, the MGMT promoter was methylated in 31 patients and unmethylated in 49 patients. In 71 patients (89%), the MGMT promoter methylation status of the primary tumor was retained at recurrence. MGMT promoter methylation, but not MGMT protein expression, was associated with longer progression-free survival, overall survival and postrecurrence survival (PRS). Moreover, PRS was increased under salvage chemotherapy. Investigation of primary and recurrent glioblastomas of 43 patients did not identify promoter methylation in any of the four MMR genes. However, recurrent glioblastomas demonstrated significantly lower MSH2, MSH6 and PMS2 protein expression as detected by immunohistochemistry. In conclusion, reduced expression of MMR proteins, but not changes in MGMT promoter methylation, is characteristic of glioblastomas recurring after the current standards of care.