Methylation of MGMT promoter as a prognostic marker of glioblastoma

Introduction. Glioblastoma is the most common malignant brain tumor in adults with a poor prognosis. Treatment of patients includes surgical resection, radiation and the alkylating agent temozolomide (TMZ). The therapeutic efficacy of TMZ is due to its ability to damage DNA and induce apoptosis, but it is neutralized by the expression of the DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT). Methylation of the MGMT gene promoter suppresses the synthesis of the corresponding enzyme and increases the cytotoxic efficiency of TMZ.
Aim. To determine the MGMT promoter methylation in glioblastoma patients and to evaluate the prognostic significance of this phenomenon.
Materials and methods. Bisulfite-treated DNA samples isolated from formalin-fixed paraffin-embedded tumor tissues obtained from glioblastoma patients were analyzed. MGMT methylation was assessed by qualitative methylation-specific polymerase chain reaction. The prognostic significance of this phenomenon in conjunction with a number of other clinical parameters was assessed by means of univariate and multivariate analysis.
Results. MGMT promoter methylation was found to be one of the most significant favorable prognostic factors of glioblastoma: the likelihood of disease reappearance or a fatal outcome at a specific point in time is approximately two-fold lower in such patients than in those with intact MGMT.
Conclusion. The methylation-specific polymerase chain reaction, which is routinely used in clinical practice, adequately assesses MGMT methylation status as a prognostic factor, but it does not allow for the evaluation of its predictive potential.

1. Gibson D., Vo A.H., Lambing H. et al. A systematic review of high impact CpG sites and regions for MGMT methylation in glioblastoma [A systematic review of MGMT methylation in GBM]. BMC Neurol 2024(1);24:103. DOI: 10.1186/s12883-024-03605-3

2. Chiesa S., Mangraviti A., Martini M. et al. Clinical and NGS predictors of response to regorafenib in recurrent glioblastoma. Sci Rep 2022;12(1):16265. DOI: 10.1038/s41598-022-20417-y

3. Brandner S., McAleenan A., Kelly C. et al. MGMT promoter methylation testing to predict overall survival in people with glioblastoma treated with temozolomide: a comprehensive metaanalysis based on a Cochrane Systematic Review. Neuro Oncol 2021;23(9):1457–69. DOI: 10.1093/neuonc/noab105

4. Esteller M., Garcia-Foncillas J., Andion E. et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 2000;343(19):1350–4. DOI: 10.1056/NEJM200011093431901

5. Hegi M.E., Diserens A.C., Gorlia T. et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 2005;352(10):997–1003. DOI: 10.1056/NEJMoa043331

6. Wick A., Kessler T., Platten M. et al. Superiority of temozolomide over radiotherapy for elderly patients with RTK II methylation class, MGMT promoter methylated malignant astrocytoma. Neuro Oncol 2020;2298:1162–72. DOI: 10.1093/neuonc/noaa033

7. Sepulveda A.R., Jones D., Ogino S. et al. CpG methylation analysis – current status of clinical assays and potential applications in molecular diagnostics: a report of the Association for Molecular Pathology. J Mol Diagn 2009;11(4):266–78. DOI: 10.2353/jmoldx.2009.080125

8. Quillien V., Lavenu A., Karayan-Tapon L. et al. Comparative assessment of 5 methods (methylation-specific polymerase chain reaction, MethyLight, pyrosequencing, methylation-sensitive highresolution melting, and immunohistochemistry) to analyze O6-methylguanine-DNA-methyltranferase in a series of 100 glioblastoma patients. Cancer 2012;118(17):4201–11. DOI: 10.1002/cncr.27392

9. Bienkowski M., Berghoff A.S., Marosi C. et al. Clinical Neuropathology practice guide 5-2015: MGMT methylation pyrosequencing in glioblastoma: unresolved issues and open questions. Clin Neuropathol 2015;34(5):250–7. DOI: 10.5414/np300904

10. Chai R.C., Liu Y.Q., Zhang K.N. et al. A novel analytical model of MGMT methylation pyrosequencing offers improved predictive performance in patients with gliomas. Mod Pathol 2019;32(1):4–15. DOI: 10.1038/s41379-018-0143-2

11. Estival A., Sanz C., Ramirez J.L. et al. Pyrosequencing versus methylation-specific PCR for assessment of MGMT methylation in tumor and blood samples of glioblastoma patients. Sci Rep 2019; 9(1):11125. DOI: 10.1038/s41598-019-47642-2

12. Yamashita S., Yokogami K., Matsumoto F. et al. MGMT promoter methylation in patients with glioblastoma: is methylation-sensitive high-resolution melting superior to methylation-sensitive polymerase chain reaction assay? J Neurosurg 2019;130(3):780–8. DOI: 10.3171/2017.11.JNS171710

13. Filipits M., Preusser M., Hainfellner J.A. et al. Evaluation of an assay for MGMT gene promoter methylation in glioblastoma samples. Anticancer Res 2020;40:6229–36. DOI: 10.21873/anticanres.14643

14. Malmstrom A., Lysiak M., Kristensen B.W. et al. Do we really know who has an MGMT methylated glioma? Results of an international survey regarding use of MGMT analyses for glioma. Neurooncol Pract 2020;7(1):68–76. DOI: 10.1093/nop/npz039

15. McAleenan A., Kelly C., Spiga F. et al. Prognostic value of test(s) for O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation for predicting overall survival in people with glioblastoma treated with temozolomide. Cochrane Database Syst Rev 2021;3(3):CD013316. DOI: 10.1002/14651858.CD013316.pub2

16. Lhotska H., Janeckova K., Cechova H. et al. Validating a clinically based MS-MLPA threshold through comparison with Sanger sequencing in glioblastoma patients. Clin Epigenetics 2025;17(1):16. DOI: 10.1186/s13148-025-01822-2

17. Tabakov D.V., Stroganova A.M., Senderovich A.I. et al. Analysis of MGMT gene methylation in gliomas by modified methyl-specific PCR. Vestnik RONTS im. N.N. Blohina = Bulletin of the N.N. Blokhin Russian Scientific Research Center 2015;26:29–33. (In Russ.).

18. Tabakov D.V., Katargin A.N., Stroganova A.M. et al. Isocitrate dehydrogenase 1 and 2 genes mutations and MGMT methylation in gliomas. Uspekhi molekulyarnoy onkologii = Advances in Molecular Oncology 2017;4(1):53–9. (In Russ.). DOI: 10.17650/2313-805X-2017-4-1-53-59

19. Pum J.K. W. Evaluation of analytical performance of qualitative and semi-quantitative assays in the clinical laboratory. Clinica Chimica Acta 2019;497:197–203. DOI: 10.1016/j.cca.2019.07.018

20. Budczies J., Klauschen F., Sinn B.V. et al. Cutoff finder: a comprehensive and straightforward web application enabling rapid biomarker cutoff optimization. PLoS One 2012;7(12):e51862. DOI: 10.1371/journal.pone.0051862

21. Gomes I., Moreno D.A., Dos Reis M.B. et al. Low MGMT digital expression is associated with a better outcome of IDH1 wildtype glioblastomas treated with temozolomide. J Neurooncol 2021;151(2):135–44. DOI: 10.1007/s11060-020-03675-6

22. Silva F.F. V. E., Di D.M., Caponio V.C. A. et al. Pyrosequencing analysis of O6-methylguanine-DNA methyltransferase methylation at different cut-offs of positivity associated with treatment response and diseasespecific survival in isocitrate dehydrogenase-wildtype grade 4 glioblastoma. Int J Mol Sci 2024;25(1):612. DOI: 10.3390/ijms25010612 23. Nguyen N., Redfield J., Ballo M. et al. Identifying the optimal cutoff point for MGMT promoter methylation status in glioblastoma. CNS Oncol 2021;10(3):CNS74. DOI: 10.2217/cns-2021-0002