1. Introduction
Glioblastoma (GBM)—World Health Organization (WHO) grade IV diffuse glioma—represents the highly invasive and infiltrative type of primary brain tumor associated with poor prognosis and a 5.6% five-year survival rate [
1,
2,
3]. GBM is the most common type of malignant central nervous system tumor in adults (47.7%–49%) that accounts for the majority of gliomas (56.6%) according to a recent Central Brain Tumor Registry of the United States (CBTRUS) Statistical Report and EUROCARE-5 study [
4]. Comprehensive genomic characterization studies revealed an underlying complex network of different molecular aberrations which provoke GBM development through changes in major signaling pathways [
5,
6]. These studies also contributed toward defining the methylation status of the O
6-methylguanine-DNA methyltransferase (
MGMT) gene promoter as one of the most relevant prognostic markers in GBM patients [
7,
8,
9,
10,
11].
The
MGMT gene encodes a DNA-repair protein that removes cytotoxic alkyl adducts from O
6-guanine [
12]. This protein inhibits the effect of cancer treatment with alkylating agents such as nitrosoureas, tetrazines, and procarbazine that induce apoptosis in cancer cells [
12,
13,
14]. The alkylating agent Temozolomide (TMZ) was approved in 2005 by the US Food and Drug Administration (FDA) for use in the treatment of GBM [
15,
16]. TMZ is an imidazotetrazine derivative of decarbazin that induces cell cycle arrest at G2/M. In Serbia, a GBM treatment protocol that includes TMZ as adjuvant therapy was introduced in 2011 [
17,
18]. Although it was demonstrated that TMZ improves the overall survival (OS) and progression-free survival (PFS) of GBM patients, at least 50% of them do not benefit from TMZ due to treatment resistance caused by over-expression of
MGMT in GBM cells [
19,
20]. To date, the bulk of evidence suggests that epigenetic silencing of the
MGMT gene through hypermethylation of the cytidine phosphate guanosinedinucleotides (CpG) in the promoter region is associated with greater response to the TMZ treatment of GBM patients [
15,
21,
22,
23,
24].
A methylation-specific polymerase chain reaction (MSP) is one of the most commonly used methods for assessing the
MGMT methylation status in either snap-frozen GBM tissue samples or formalin-fixed, paraffin-embedded (FFPE) tissue [
25,
26,
27,
28]. This method is based on sodium bisulfite treatment of isolated DNA samples which results in the conversion of unmethylated cytosines into uracil, leaving methylated cytosines unchanged. Bisulfite conversion of template DNA is followed by PCR reactions using two primer sets for both an unmethylated and methylated
MGMT promoter variant, which allow for the evaluation of the methylation status at six to nine CpG sites [
28,
29]. The difference in amplicon lengths after conducting PCR reactions with primer sets for each variant of
MGMT promoter provides easy-to-interpret results that can be visualized by agarose gel electrophoresis. Since MSP was established, this method has evolved as the “gold standard” that enables a cost-efficient non-quantitative method of
MGMT methylation analysis suitable for routine clinical diagnostics with low sample numbers [
27].
The main goal of our study was to determine MGMT promoter methylation and its relevance for the prediction and prognosis of clinical outcomes of the Serbian population with glioblastoma. The study was designed to investigate the effect of novel therapeutic treatment (TMZ) on overall survival. Also, the potential use of MSP as a semi-quantitative method for assessing MGMT methylation status in snap-frozen GBM samples was investigated.
4. Discussion
There is ongoing debate concerning the most suitable technique for the determination of the
MGMT promoter methylation and the prognostic importance of the obtained methylation status for patients with GBM [
28,
34]. MGMT testing in our study is performed by MSP as one of the oldest and the most widely used techniques [
25,
26,
27,
28]. Notably, MSP is cost-effective, gel-based, and the most appropriate method for resource-limited settings and routine diagnostics with low sample numbers. However, this technique is especially prone to producing false-positive results when performed on low quality/quantity DNA, partially bisulfite-converted DNA, or tumor specimens with irregular mosaic methylation patterns [
28]. Generally, only vital (non-necrotic) tumor specimens should be used for MSP analysis to avoid false-negative results [
28].
In order to improve MSP semi-quantitative potentials, we performed additional normalization of the methylation signal by
ALU control and universal positive methylation control [
32,
33]. Therefore, we compensate PMR for variations in copy number due to differences in sample handling, DNA isolation and tumor heterogeneity. Optimally standardized and easy-to-interpret MSP data were used in our study for evaluation of the clinical importance of the methylation status of the
MGMT promoter.
Further, numerous GBM clinical trials with TMZ have established a positive methylation status of the
MGMT promoter as the strongest predictor for OS and progression-free survival (PFS) benefit [
13,
19,
23,
27,
35]. However, our study showed no significant impact of the
MGMT promoter methylation on the survival outcome and TMZ treatment benefit. Although, we should emphasize that these are only preliminary data based on low sample quantity. Nevertheless, the same observation was made in the above-mentioned study of 110 GBM patients from Serbia; although, the methylation status was assessed in only 62 patients (56.4%) of the cohort [
17].
Controversial observations about the predictive and prognostic value of
MGMT promoter methylation were noted in several studies [
14,
36] and in meta-analysis [
13]. Jesien-Lewandowicz et al. (2009) detected a positive methylation status in 23 out of 32 (72%) primary GBM patients from Poland treated with surgery and radiotherapy [
14]. In univariate analysis, the presence of
MGMT promoter methylation was not associated with the patient’s gender and longer survival. Kalkan and colleagues (2015) assessed
MGMT promoter methylation status on 40 primary glioblastoma from Turkish patients [
36]. They found positive methylation in 13 samples (32.5%) and no statistical significance between
MGMT methylation and gender and overall survival.
Intratumoral and temporal heterogeneity may underlie the described discrepancies in our and other studies with negative prognostic values of the
MGMT status [
37]. Alternatively, negative conclusions in
MGMT studies with Polish, Turkish, and Serbian GBM patients may reflect population molecular differences in gliomagenesis. Although, we should mention that these are small size studies which require confirmation in larger-scale, prospective controlled trials. Previously, Wiencke et al. (2005) showed a substantial ethnic specificity of molecular features (
MGMT,
TP53 and
EGFR) in 556 glioma samples in the San Francisco Bay Area [
38].
Our study has several limitations. First, it was conducted on small cohorts of patients from a single Clinical Centre in Serbia and the obtained results should be interpreted with care. Therefore, we could not definitively rule out the prognostic value of the
MGMT promoter methylation status in the Serbian GBM population. Second, only the independent prognostic value of
MGMT methylation was considered. Although the study was carefully performed, the complexity of gliomagenesis and the latest WHO classification of glioma 2016, suggested that the combination of
MGMT,
IDH1, and/or
TP53 analysis is more relevant for the prediction of survival of patients with GBM [
2].
The significance of the combination of predictive biomarkers rather than their individual status for survival prediction in patients with GBM was demonstrated widely [
39,
40,
41,
42]. Meta-analysis of Zou and colleagues suggested that
IDH mutations were tightly associated with
MGMT promoter hypermethylation (
p < 0.001) and
TP53 gene mutation (
p < 0.001) [
39]. They indicated that the
IDH mutation rate was linked to the glioma’s genomic profile. Higher rates of G to A transitions in
IDH1 codon 132 and
TP53 codons 248 and 273 were explained by higher levels of methylation of the
MGMT promoter CpG islands [
39,
40]. These mutational events were considered as early events in gliomagenesis which might affect a common stem glial precursor cell population. They were linked with a low proliferation tumor phenotype and a favorable prognosis in glioma patients. Similarly, Shamsara et al. (2009) detected hypermethylation of the
MGMT promoter in 24 out of 50 patients (48%) and mutation of
TP53 gene in 26 out of 50 patients (52%) with primary glioblastoma in Iran [
41]. A significant association between
MGMT methylation status and
TP53 mutation status was found (
p < 0.05).
TP53 mutations were observed in 17 out of 26 patients (65.4%) with
MGMT-hypermethylated glioblastoma. Likewise, in the previously mentioned study of Jesien-Lewandowicz and associates, the frequency of TP53 G:C to A:T mutations were higher in patients with
MGMT promoter methylation (6 out of 23 patients (26%),
p = 0.376) [
14]. Further, Wang et al. (2014) investigated the predictive value of the combination of
MGMT methylation status and
TP53 and
IDH1 mutation status in 78 patients with GBM from China [
42]. For patients with
IDH1 mutation,
MGMT hypermethylation was correlated with better overall survival (
p = 0.013), while for the patients without
IDH1 mutation, the presence of
TP53 mutation was associated with improved survival (
p = 0.029).
A remarkable improvement in the overall survival of GBM patients is recorded from 2005 since the approval of TMZ for concomitant treatment with radiotherapy (RT) and adjuvant treatment for newly diagnosed GBM [
15,
22,
24]. Meta-analysis of survival outcomes of newly diagnosed GBM patients revealed that the RT + TMZ-treated group of patients had a significantly higher median survival (13.41–19 months) in comparison with RT-alone group (7.7–17.1 months) [
22].
In Serbia, TMZ was introduced in 2011. Recent studies suggested that TMZ treatment had a favorable impact on the overall survival of GBM patients in Serbia [
17,
18]. In comparison with RT + BCNU/CCNU treatment, the overall survival of TMZ treated patients was significantly higher (the first study 19 months vs. 13 months; the second study 14.79 months vs. 9. 91 months) [
17,
18]. Our study confirmed previous findings regarding the favorable impact of TMZ treatment on OS of GBM patients in Serbia (15 months vs. 5 months).