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Running title: Local temozolomide in glioma patients 4
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Local delivery of temozolomide via a biologically inert carrier (Temodex) prolongs survival of 6
glioma patients irrespectively of the MGMT methylation status. 7
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KARLSSON I1*, VEEVNIK D2, FEDULOV A3, YURKSHTOVICH N4, YURKSHTOVICH T4, 9
PEJLER, G5,6, LOKOT I1 10
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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
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*Corresponde nce: iulia@doublebp.com 19
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Received June 13, 2018 / Accepted August 28, 2018 21
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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
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Key words: glioma, Temodex, temozolomide, MGTM, methylation 39
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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
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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, http://rceth.by/Refbank/reestr_lekarstvennih_sredstv/results). 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
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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
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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
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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
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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
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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
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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
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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
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Acknowledgements 207
We are grateful to Emergenc y Hospital #5 (Minsk, Belarus) for providing samples for this study. 208
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Disclosures 210
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IK and IL are emplo yed by Double Bond Pha rmaceutical. GP receives consulting fees from Double 211
Bond Pharmaceutical. 212
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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
282
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
288
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
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Table 1. Clinical and demographic characteristics. 296
Study group
(n=29)
Control group
(n=49)
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