Glioblastoma (GBM) is the most common type of brain tumor, with high mortality rates. The aggressive biology of GBM and resistance to therapy are major clinical problems for patients diagnosed with the disease (1). At present, standard treatment includes maximal safe surgery, concomitant radiotherapy and temozolomide (TMZ) chemotherapy, where a median survival time of <15 months is observed in patients fit for this current first-line therapy (1). In terms of years of life lost, the population burden from GBM is the highest of all malignancies (2). GBM frequently affects young people (30–40 years of age), as well as the elderly (3). Physically or cognitively disabled patients require care from the early stages of the disease, which results in increased expenses. Despite advances in therapeutic strategies, no significant reductions in the age-standardised incidence and mortality rates of this disease have been observed within the last decade (3).

Patients with a good Eastern Cooperative Oncology Group performance status or Karnofsky score at the time of diagnosis undergo surgical resection (4,5). Complete removal of the tumor is challenging due to the infiltrative nature of GBM; however, 80–95% debulking is typically achieved unless the disease is in a neurologically eloquent location (6). The standard treatment, known as ‘The Stupp Protocol’, consists of concurrent temozolomide (TMZ) therapy and radiotherapy (60 Gy/30 fractions), followed by subsequent adjuvant TMZ therapy for 6–12 cycles (1). TMZ functions by alkylating the guanine residues in DNA, resulting in mispairing with thymine during DNA replication and cellular arrest in rapidly dividing cells (7,8).

Methylguanine-DNA methyltransferase (MGMT) removes mutagenic alkyl adducts and, thus, protects DNA from the damage induced by TMZ and other alkylating agents. Loss of MGMT expression commonly occurs in human neoplasia (9). The expression of MGMT is regulated by hypermethylation of the CpG islands within the promoter and enhancer regions of the gene (10–12). MGMT deficiency is presumed in GBMs with a methylated MGMT promoter, resulting in the enhanced cytotoxicity of TMZ (13). Studies have demonstrated that presence of MGMT promoter methylation is correlated with the response of patients to treatment with TMZ; thus, it is considered to be a predictive biomarker in TMZ-treated GBMs, even in elderly patients (14,15). Between 25 and 50% of GBMs are methylated at the MGMT promoter; therefore, using MGMT methylation status clinically as a predictive biomarker for GBM patients has received increased attention, with the long-term aim to use it as a biomarker to assign alkylating therapy to individual patients. However, a number of studies have reported discordance between MGMT methylation status and treatment outcome, and no clear alternative treatment is available for patients with unmethylated MGMT tumors, who derive only limited benefit from TMZ (13).

Based on the signatory gene expression profiles using data generated from The Cancer Genome Atlas (TCGA) (16), four distinct molecular subtypes of GBM were described: Classical, mesenchymal, neural and proneural (17). These subtypes have also been associated with characteristic genetic alterations (17). The classical subtype is associated with an astrocytic expression profile, with frequent epidermal growth factor receptor (EGFR) amplification, concomitant chromosome 7 amplification and chromosome 10 loss, and focal deletions of 9p encompassing cyclin-dependent kinase inhibitor 2A (CDKN2A). Notably, the classical subtype is not associated with TP53 mutations, which are common in GBMs (17). The mesenchymal subtype is typified by the expression of mesenchymal markers, with frequent deletions or mutations of the neurofibromin 1 (NF1) and phosphatase and tensin homolog (PTEN) genes. The neural subtype exhibits expression of neuronal markers and displays various mutations and copy number alterations, including amplification of EGFR and deletion of PTEN. The proneural subtype exhibits an oligodendrocytic expression signature and is characterized by TP53 mutations. In addition, focal amplifications of the chromosome 4q12 region, which contains the oligodendrocytic development gene α-type platelet-derived growth factor receptor (PDGFRA), or mutations of the isocitrate dehydrogenase 1 gene (IDH1) are observed. The TCGA group has recently published an updated comprehensive genomic, epigenomic, transcriptomic and proteomic analysis of >500 GBMs (18). Patients were categorized into the four different groups and the overall survival was stratified based on MGMT methylation. Notably, the findings indicated that MGMT methylation may be a predictive biomarker for response to treatment, but only in the classical subtype of GBM.

In the present study, MGMT methylation and somatic mutations were assessed using semiconductor sequencing technology in the same tumor DNA. The cohort included biopsies from 35 GBM patients, who had been treated uniformly with standard concurrent radiotherapy plus TMZ. The association between MGMT methylation status/mutation profile and survival was also evaluated.

The presence of MGMT promoter methylation in GBM is a predictor of response to TMZ treatment and of overall survival. In the present study, the mutations detected in MGMT methylated GBMs were four times higher compared with MGMT unmethylated GBMs. This observation may be attributed to a lack of DNA repair function upon presence of lower levels of the MGMT protein.

In 2005, MGMT promoter methylation was reported to be associated with a survival benefit from alkylating agent chemotherapy in GBM patients, in a companion study based on a phase III concurrent radiation therapy and TMZ trial (13). A number of studies have demonstrated an even higher survival benefit in patients treated with alkylating agents, who presented tumors with MGMT promoter methylation, compared with patients having tumors with an unmethylated MGMT promoter (13,22,23). Therefore, MGMT promoter methylation is a favorable prognostic marker, in addition to a favorable predictive marker for response to TMZ treatment in patients with GBM. The molecular basis for the differential response to TMZ treatment in patients is well established. Alkylating drugs, including TMZ, induce DNA damage by introducing alkyl adducts into the DNA molecules. This causes genetic mutations and cross-links between DNA strands that inhibit DNA replication and thereby trigger cell death. However, MGMT removes the alkyl adducts from the DNA as they are introduced, preventing subsequent mutational damage. Tumor cells expressing MGMT are thus resistant to alkylating drugs (24).

In the current study, the TTP following chemoradiotherapy and subsequent overall survival were significantly increased in patients with MGMT methylation compared with patients without methylated tumors. Thus, the present study confirmed the role of MGMT methylation and its associated sensitivity to TMZ (25,26).

The current study investigated the prevalence and types of genomic alterations in GBM and whether these correlate with MGMT methylation. The results demonstrated that 75% of MGMT methylated tumors harbored one or more somatic mutations, compared with 35% of unmethylated cases. Overall, the mutations identified in MGMT methylated were four times higher compared with MGMT unmethylated tumors. Notably, mutations were identified in the TP53, CDKN2A, PTEN and PIK3CA genes in MGMT methylated cases. Since these cases also exhibited improved overall survival, further investigations should determine whether these genomic aberrations work synergistically with MGMT methylation status and contribute to the response to treatment and overall prognosis. MGMT methylation may play a dual role, increasing sensitivity to TMZ and increasing genetic instability, particularly by facilitating the appearance of G to A transition mutations. The TCGA dataset of GBMs revealed increased frequencies of TP53 and PTEN point mutations in MGMT methylated GBMs compared with MGMT unmethylated GBMs (16). In the current study, mutations in PTEN and TP53 were also found to be associated with MGMT promoter methylation, confirming the findings of the TCGA. MGMT methylation is commonly associated with G:C to A:T mutations in KRAS and TP53 genes in colorectal cancer (27,28). Therefore, MGMT promoter methylation may be an early event in the development of methylated GBM, preceding other characteristic mutations in this subgroup.

In the present study, higher frequencies of EGFR and CDKN2A alterations were observed in the MGMT methylation group. Although the tumors were not categorized into proneural, neural, mesenchymal or classical subtypes (17), the higher frequencies of EGFR/CDKN2A indicate that the MGMT methylation group may be enriched for the classical subtype. Furhtermore, the current study is in accordance with the findings of Brennan et al (18), demonstrating that the classical subtype is more responsive to TMZ, which is reflected by the longer TTP and overall survival observed.

In conclusion, MGMT methylation was strongly associated with the response to TMZ treatment and overall survival in GBMs, as previously reported. Methylation was also found to be associated with a significantly higher mutation rate. Therefore, it is hypothesized that methylation may be one of the earliest preneoplastic events in this subgroup. A larger cohort of patients is required to further investigate the association of MGMT methylation and specific mutations (including those in EGFR, CDKN2A and TP53) with overall survival in TMZ-treated GBMs.