MGMT Promoter Methylation in Glioma: ESMO Biomarker Factsheet

MGMT in cancer

The O⁶-methylguanine-DNA methyl-transferase (MGMT) gene is located on chromosome 10q26.3, and encodes a highly evolutionarily conserved and ubiquitously expressed enzyme involved in DNA repair [1]. MGMT acts by removing alkyl adducts from the O⁶ position of guanine at DNA level, thus antagonizing the lethal effects of alkylating agents. During the repair process, the methyl moiety of the O⁶-methylguanine adduct is transferred to the MGMT protein, which subsequently undergoes irreversible inhibition. By contrast, defective MGMT function results into persistence of the O⁶-methylguanine adduct, which causes base misrepairing and mismatch repair futile cycling during DNA replication, eventually leading to cell cycle arrest and apoptosis. Importantly, epigenetic modification could silence the MGMT gene,commonly through methylation of the cytosine-phosphate-guanine (CpG) island at specific CpG sites within the MGMT gene promoter. Therefore, MGMT gene promoter methylation induces loss/low levels of functional MGMT protein, thus producing inadequate repair of DNA alkylation in response to alkylating chemotherapy [2].

MGMT in glioma

Methylation of the MGMT gene promoter has been observed in approximately 50% of grade IV gliomas, commonly referred to as glioblastoma multiforme (GBM) [1]. Remarkably, in cases with monosomy of chromosome 10, a common event in GBMs, methylation of the remaining allele completely blocks MGMT-mediated DNA repair [2].

Given its relatively high frequency in GBM, which may vary based on the method that is used for its assessment, MGMT gene promoter methylation has been investigated as a potential biomarker of sensitivity to alkylating chemotherapy, including temozolomide (TMZ). Originally, the first evidence that associated MGMT methylation status and response to TMZ emerged from the ‘Stupp trial’ [3]. This study established a new standard of care based on chemo-radiotherapy following surgical management for newly diagnosed GBMs, as it showed the superiority of TMZ given concomitantly with radiotherapy, and then sequentially as single agent for up to six cycles versus radiotherapy alone [4].

MGMT as a prognostic biomarker

In the ‘Stupp trial’ MGMT gene promoter methylation was found to be the strongest predictor of survival regardless of whether GBM patients were treated with TMZ plus radiotherapy or radiotherapy alone [3,5]. Although MGMT-methylated patients derived a greater benefit from the addition of TMZ to radiotherapy as compared to MGMT-unmethylated GBMs, the ‘Stupp trial’ was not powered to show statistical significance for subgroup analysis according to MGMT status, and the positive interaction between treatment with TMZ and MGMT gene promoter methylation was more hypothesis-generating rather than conclusive.

Clear evidence on the prognostic role of MGMT gene promoter methylation came from the RTOG0525 trial, in which newly diagnosed GBM patients were randomised to two different sequential treatments of TMZ after completion of standard TMZ with concurrent radiotherapy [6]. In this trial, sequential treatment was assigned according to MGMT status: MGMT-methylated GBMs received standard maintenance therapy as in the ‘Stupp trial’ (TMZ for five consecutive days every 28 days), while MGMT-unmethylated patients received an intensive maintenance schedule (lower-dose of TMZ for 21 consecutive days every 28 days). Importantly, the MGMT status was found to be a strong prognostic factor as the median survival was 21.2 months in MGMT- methylated GBMs versus 14.0months in unmethylated.

MGMT as a predictive biomarker

In 2005, the ‘Stupp trial’ reported that the survival benefit observed with the addition of TMZ to standard radiotherapy was much larger in MGMT-methylated GBMs as compared to patients with MGMT-unmethylated tumours [3]. Since then, long-term results of this trial have been published, which confirmed the potential value of MGMT gene promoter methylation in predicting a favourable response to TMZ(table 1).

The predictive role of MGMT status was definitely assessed by the ‘NOA-08’ and “Nordic Elderly” randomised trials, which compared TMZ monotherapy to radiotherapy alone in elderly GBM patients (≥ 65 years and > 60 years, respectively) [7,8]. In both trials a longer survival was observed in MGMT-methylated GBMs treated with TMZ monotherapy in comparison with MGMT-unmethylated patients. By contrast, outcome did not differ significantly between patients in the radiotherapy arm according to MGMT status. More recent studies have shown that withholding TMZ in MGMT-unmethylated patients in favour of an alternative treatment not including TMZ does not appear to be detrimental in terms of overall survival, further confirming the value of MGMT gene promoter methylation as predictor of sensitivity to TMZ [9,10].

However, a few issues still prevent the implementation of the MGMT parameter into routine clinical decision-making. First, MGMT gene promoter methylation holds strong prognostic value, which might have confounded its role as pure predictive factor of response to TMZ [3,5-8]. Secondly, a slight benefit in favour of TMZ plus radiotherapy versus radiotherapy alone was observed in the MGMT-unmethylated patients of the ‘Stupp trial’, as well as in another trial of elderly patients (≥ 65 years) with MGMT-unmethylated GBMs treated with TMZ plus short-course radiation versus short-course radiation alone, which suggests that GBMs patients may occasionally benefit from TMZ chemotherapy even in the absence of MGMT gene promoter methylation [3,5,11]. For this reason, at this time MGMT status does not have an impact on the decision to offer standard treatment with radiation plus alkylating chemotherapy in newly diagnosed GBMs, which means that GBM patients, when appropriate, are all managed the same with TMZ plus radiotherapy.

MGMT testing recommendations

To date, no large consensus exists on the optimal method of assessment of MGMT gene promoter methylation, and different assays are available for this purpose. Regardless of the type of the chosen technique, the preliminary phases consist of genomic DNA extraction, its qualitative/quantitative check, and bisulfite conversion. As bisulfite changes cytosine residues in thymine, this treatment allows to distinguish the presence of methylated or unmethylated sequences at MGMT gene promoter only if the nucleotide is unbound to DNA adduct.

The simple Methylation Specific-PCR (MS-PCR), and the Methylation Sensitive–quantitative Locked Nucleic Acid PCR (MS-qLNAPCR) are the two most commonly used methods, as both are supported by evidence coming from several randomised trials [2]. SimpleMS-PCR is a reaction that allows to selectively amplify the methylated/unmethylated MGMT gene promoter components for the same DNA sample. The characterisation is mainly qualitative, in fact the PCR products are separated by electrophoresis and visualised by transilluminator as bands. However, based on intensity of bands, it is also possible to carry out a semi-quantitative analysis. MS-PCR is a highly standardised technique; the results are solid, reliable and are obtained in a few hours. However, this technique does not allow a precise quantitative evaluation of the methylated/unmethylated MGMT gene promoter components and the sensibility of detection is lower than the MS-qLNAPCR. For this reason, it is possible that small cellular clones with methylated MGMT gene promoter are missed. For qualitative assessments of MGMT gene promoter methylation, a two-step nested MS-PCR employing a Taq Gold polymerase kit is done [12]. Amplicons are then separated by SeaKem LE agarose gel, purified, and sequenced by Sanger’s method.

Quantitative MS-qLNAPCR is a real time PCR analysis that shares the same biological bases of MS-PCR with a few technical differences. MS-qLNAPCR analysis requires a proven and solid technical experience of the operators as well as specific instruments and reagents, which increases the costs for each patient. The amplification of methylated/unmethylated MGMT gene promoter is done by PCR but its detection is obtained using fluorophores. This system increases the sensitivity of the analysis, which allows the identification of small tumoural cellular clones, and reduces the number of false negative cases. For each sample a double reaction must be set up in order to reduce the analytic bias and guarantee reproducible results. Briefly, Real Time PCR analysis, is performed using bisulfite treated DNA, FastStartTaq™ polymerase, locked nucleic acid modified primers, one for the methylated and one for the unmethylated sequence, and beacon probes, recognising the methylated and unmethylated alleles. The SNURF gene promoter methylation is usually used as a reference for normalisation [13]. The presence of a cytosine residue after bisulfite treatment indicates that the cytosine residue is protected by methylation from bisulfite modification. Relative quantification of the methylated and unmethylated allele ratio is calculated according to DDCt method using an equal amount of SssI-treated wild-type DNA (fully methylated) mixed with the same amount of untreated DNA as a calibrator [13,14].

Ensuring quality and timely testing results

The major concerns with the evaluation of MGMT gene promoter methylation are based on the absence of validation studies of available assays for its technical reproducibility. Strict quality control studies, both in processing and assessment of data, would need the collaborative effort of neuro-oncological surgeons, neuropathologists, and molecular pathologists. The methods and optimal cut-off definitions for determination of MGMT status remain controversial. Simple qualitative method such as MS-PCR is likely inferior to a quantitative assay like MS-qLNAPCR, as the first precludes the evaluation of the extent of methylation and could miss the methylation of low percentage of cells. Since even low levels of MGMT promoter methylation appear to predict sensitivity to alkylating agents, the MS-qLNAPCR assay comes out as an accurate and robust test.

In order to limit false negative and false positive results, with both MS-PCR and MS-qLNAPCR techniques, in-house quality assessment should be carried out by using internal controls.

Patient selection

Currently, consensus exists on the fact that MGMT status should be assessed in all patients with a histological diagnosis of GBM according to World Health Organization (WHO) 2016 classification, as in this context MGMT status holds strong prognostic value and potential predictive information on benefit from TMZ chemotherapy.

On one hand, MGMT-methylated patients should receive radiotherapy plus TMZ followed by TMZ chemotherapy if they meet clinically appropriate criteria. On the other, for MGMT-unmethylated patients, especially if aged 65 years or older, the choice of withholding TMZ treatment should be tailored according to the risk-benefit profile (including comorbidities and performance status) of each individual.

In grade II/III gliomas (lower-grade gliomas, LGGs), the clinical impact of MGMT status on benefit from TMZ or other alkylating agents remains to be determined. That is mainly because MGMT gene promoter methylation in LGGs is often significantly associated with other biomarkers (i.e. 1p/19q codeletion and/or IDH mutation) that are well established prognostic factors for survival [18,19]. For this reason, it is nowaday difficult to comprehend the true prognostic or predictive value of MGMT status in LGGs. As a result, determination of MGMT gene promoter methylation has no role in LGGs for selection of optimal treatment.

References

1. Wick W, Weller M, van den Bent M, et al. MGMT testing – the challenges for biomarker-based glioma treatment. Nat Rev Neurol 2014;10:372-85. 
2. Mansouri A, Hachem LD, Mansouri S, et al. MGMT promoter methylation status testing to guide therapy for glioblastoma: refining the approach based on emerging evidence and current challenges. Neuro Oncol; Published 5 September 2018. doi: 10.1093/neuonc/noy132. 
3. Hegi ME, Diserens AC, Gorlia T, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 2005; 352:997-1003.
4. Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005; 352:987-96.
5. Stupp R, Hegi ME, Mason WP, et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009; 10:459-66.
6. Gilbert MR, Wang M, Aldape KD, et al. Dose-dense temozolomide for newly diagnosed glioblastoma: a randomized phase III clinical trial. J Clin Oncol 2013; 31:4085-91.
7. Wick W, Platten M, Meisner C, et al. Temozolomide chemotherapy alone versus radiotherapy alone for malignant astrocytoma in the elderly: the NOA-08 randomised, phase 3 trial. Lancet Oncol 2012; 13:707-15.
8. Malmström A, Grønberg BH, Marosi C, et al. Temozolomide versus standard 6-week radiotherapy versus hypofractionated radiotherapy in patients older than 60 years with glioblastoma: the Nordic randomised, phase 3 trial. Lancet Oncol 2012; 13:916-26.
9. Wick W, Gorlia T, Bady P, et al. Phase II study of radiotherapy and temsirolimus versus radiochemotherapy with temozolomide in patients with newly diagnosed glioblastoma without MGMT promoter hypermethylation (EORTC 26082). Clin Cancer Res 2016; 22:4797-806.
10. Herrlinger U, Schäfer N, Steinbach JP, et al. Bevacizumab plus irinotecan versus temozolomide in newly diagnosed O6-methylguanine-DNA methyltransferase nonmethylated glioblastoma: the randomized GLARIUS trial. J Clin Oncol 2016; 34:1611-9.
11. Perry JR, Laperriere N, O'Callaghan CJ, et al. Short-course radiation plus temozolomide in elderly patients with glioblastoma. N Engl J Med 2017; 376:1027-37. 
12. Palmisano WA, Divine KK, Saccomanno G, et al. Predicting lung cancer by detecting aberrant promoter methylation in sputum. Cancer Res 2000; 60:5954-8. 
13. Morandi L, Franceschi E, de Biase D, et al. Promoter methylation analysis of O6-methylguanine-DNA methyltransferase in glioblastoma: detection by locked nucleic acid based quantitative PCR using an imprinted gene (SNURF) as a reference. BMC Cancer 2010; 10:48.
14. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25:402-8.
15. Leu S, von Felten S, Frank S, et al.IDH/MGMT-driven molecular classification of low-grade glioma is a strong predictor for long-term survival. Neuro Oncol 2013; 15:469-79.
16. Wick W, Meisner C, Hentschel B, et al. Prognostic or predictive value of MGMT promoter methylation in gliomas depends on IDH1 mutation. Neurology 2013; 81:1515-22. 
17. Bell EH, Zhang P, Fisher BJ, et al. Association of MGMT Promoter Methylation Status With Survival Outcomes in Patients With High-Risk Glioma Treated With Radiotherapy and Temozolomide: An Analysis From the NRG Oncology/RTOG 0424 Trial. JAMA Oncol 2018; 4:1405-9. 
18. Xia L, Wu B, Fu Z, et al. Prognostic role of IDH mutations in gliomas: a meta-analysis of 55 observational studies. Oncotarget 2015; 6:17354-65. 
19. Zhao J, Ma W, Zhao H. Loss of heterozygosity 1p/19q and survival in glioma: a meta-analysis. Neuro Oncol 2014; 16:103-12.