Journal of Neuro-Oncology

, Volume 140, Issue 3, pp 559–568 | Cite as

Defining a prognostic score based on O6-methylguanine-DNA methyltransferase cut-off methylation level determined by pyrosequencing in patients with glioblastoma multiforme

  • Elisa De Carlo
  • Lorenzo Gerratana
  • Giovanna De Maglio
  • Vanessa Buoro
  • Francesco Cortiula
  • Lorena Gurrieri
  • Miriam Isola
  • Gianpiero Fasola
  • Fabio Puglisi
  • Stefano Pizzolitto
  • Simona Rizzato
Clinical Study



Epigenetic variations in the O6-methylguanine-methyltransferase gene had been widely associated with a favorable impact on survival in patients affected by glioblastoma multiforme (GBM). Aim of this study is to explore a scoring system based on the gene promoter methylation in order to predict patients’ prognosis.


A series of 128 patients with GBM was retrospectively analyzed. A training set and a validations set were then generated. The methylation level of CpGi from 74 to 83 was determined by pyrosequencing. In accordance to previous literature, each island was assigned with 1 point if the corresponding methylation level was higher than 9%. The sum consisted in a score that went from 0 (all CpGi < 9%) to 10 (all CpGi ≥ 9%). A threshold capable to detect a favorable outcome (overall survival, OS > 24 months) was identified by ROC analysis.


Median OS and follow-up were 14 and 32.6 months respectively. Among the total population, 35% of the pts had a score of 0, while 29% had a score of 10. A score ≥ 6 was associated with a favorable prognosis also when corrected for age (> 70 vs. ≤ 70 years) and ECOG performance status (0–1 vs. 2–3). Similar results were observed also in terms of PFS. Results were consistent in the training and in the validation set.


The present manuscript explored a novel scoring system capable to take into consideration the methylation status of each single CpGi, capable to better predict prognosis in GBM patients.


Glioblastoma multiforme Prognostic score MGMT cut-off methylation level CpG islands 



No person other than the authors contributed to this work.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study, whenever possible, taking in account the retrospective nature of the study.


  1. 1.
    Louis DN, Ohgaki H, Wiestler OD et al (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114:97–109CrossRefGoogle Scholar
  2. 2.
    Minniti G, De Sanctis V, Muni R et al (2008) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma in elderly patients. J Neurooncol 88:97–103CrossRefGoogle Scholar
  3. 3.
    Jhanwar-Uniyal M, Labagnara M, Friedman M et al (2015) Glioblastoma: molecular pathways, stem cells and therapeutic targets. Cancers 7:538–555CrossRefGoogle Scholar
  4. 4.
    Johnson DR, O’Neill BP (2012) Glioblastoma survival in the United States before and during the temozolomide era. J Neurooncol 107:359–364CrossRefGoogle Scholar
  5. 5.
    Hegi ME, Liu L, Herman JG et al (2008) Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity. J Clin Oncol 26:4189–4199CrossRefGoogle Scholar
  6. 6.
    Esteller M, Garcia-Foncillas J, Andion E et al (2000) Inactivation of the DNA-repair Gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 343:1350–1354CrossRefGoogle Scholar
  7. 7.
    Hegi ME, Diserens AC, Godard S et al (2004) Clinical trial substantiates the predictive value of O-6-methylguanine- methyltransferase promoter methylation in glioblastoma patients treated with temozolomide. Clin Cancer Res 10:1871–1874CrossRefGoogle Scholar
  8. 8.
    Hegi ME, Diserens A-C, Gorlia T et al (2005) MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352:997–1003CrossRefGoogle Scholar
  9. 9.
    Gorlia T, van den Bent MJ, Hegi ME et al (2008) Nomograms for predicting survival of patients with newly diagnosed glioblastoma: prognostic factor analysis of EORTC and NCIC trial 26981–22981/CE.3. Lancet Oncol 9:29–38CrossRefGoogle Scholar
  10. 10.
    Pegg AE (1990) Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogens and therapeutic agents. Cancer Res 50:6119–6129Google Scholar
  11. 11.
    Esteller M (2002) CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene 21:5427–5440CrossRefGoogle Scholar
  12. 12.
    Brandes AA, Franceschi E, Tosoni A et al (2009) Temozolomide concomitant and adjuvant to radiotherapy in elderly patients with glioblastoma: correlation with MGMT promoter methylation status. Cancer 115:3512–3518CrossRefGoogle Scholar
  13. 13.
    Malmström A, Grønberg BH, Marosi C et al (2012) 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 13:916–926CrossRefGoogle Scholar
  14. 14.
    Wick W, Platten M, Meisner C et al (2012) Temozolomide chemotherapy alone versus radiotherapy alone for malignant astrocytoma in the elderly: the NOA-08 randomised, phase 3 trial. Lancet Oncol 13:707–715CrossRefGoogle Scholar
  15. 15.
    Mikeska T, Bock C, EI-Maarri O et al (2007) Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis. J Mol Diagn 9:368–381CrossRefGoogle Scholar
  16. 16.
    Karayan-Tapon L, Quillien V, Guilhot J et al (2010) Prognostic value of O6-methylguanine-DNA methyltransferase status in glioblastoma patients, assessed by five different methods. J Neurooncol 97:311–322CrossRefGoogle Scholar
  17. 17.
    Quillien V, Lavenu A, Karayan-Tapon L et al (2012) Comparative assessment of 5 methods (methylation-specific polymerase chain reaction, methylight, pyrosequencing, methylation-sensitive high-resolution melting, and immunohistochemistry) to analyze O6-methylguanine-DNA- methyltranferase in a series of 100. Cancer 118:4201–4211CrossRefGoogle Scholar
  18. 18.
    Preusser M, Berghoff AS, Manzl C et al (2014) Clinical neuropathology practice news 1-2014: pyrosequencing meets clinical and analytical performance criteria for routine testing of MGMT promoter methylation status in glioblastoma. Clin Neuropathol 33:6–14CrossRefGoogle Scholar
  19. 19.
    Quillien V, Lavenu A, Ducray F, et al (2016) Validation of the high-performance of pyrosequencing for clinical MGMT testing on a cohort of glioblastoma patients from a prospective dedicated multicentric trial. Oncotarget 7:61916–61929CrossRefGoogle Scholar
  20. 20.
    Weller M, Stupp R, Reifenberger G et al (2010) MGMT promoter methylation in malignant gliomas: ready for personalized medicine? Nat Rev Neurol 6:39–51CrossRefGoogle Scholar
  21. 21.
    Felsberg J, Thon N, Eigenbrod S et al (2011) Promoter methylation and expression of MGMT and the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 in paired primary and recurrent glioblastomas. Int J Cancer 129:659–670CrossRefGoogle Scholar
  22. 22.
    Reifenberger G, Hentschel B, Felsberg J et al (2012) Predictive impact of MGMT promoter methylation in glioblastoma of the elderly. Int J Cancer 131:1342–1350CrossRefGoogle Scholar
  23. 23.
    Dunn J, Baborie A, Alam F et al (2009) Extent of MGMT promoter methylation correlates with outcome in glioblastomas given temozolomide and radiotherapy. Br J Cancer 101:124–131CrossRefGoogle Scholar
  24. 24.
    Brigliadori G, Foca F, Dall’Agata M et al (2016) Defining the cutoff value of MGMT gene promoter methylation and its predictive capacity in glioblastoma. J Neurooncol 128:333–339CrossRefGoogle Scholar
  25. 25.
    Gurrieri L, De Carlo E, Gerratana L et al (2018) MGMT pyrosequencing-based cut-off methylation level and clinical outcome in patients with glioblastoma multiforme. Future Oncol 14:699–707CrossRefGoogle Scholar
  26. 26.
    Yan H, Parsons DW, Jin G et al (2009) IDH1 and IDH2 mutations in gliomas. N Engl J Med 360:765–773CrossRefGoogle Scholar
  27. 27.
    Wick W, Hartmann C, Engel C et al (2009) NOA-04 randomized phase III trial of sequential radiochemotherapy of anaplastic glioma with procarbazine, lomustine, and vincristine or temozolomide. J Clin Oncol 27:5874–5880CrossRefGoogle Scholar
  28. 28.
    Van Den Bent MJ, Dubbink HJ, Marie Y et al (2010) IDH1 and IDH2 mutations are prognostic but not predictive for outcome in anaplastic oligodendroglial tumors: a report of the European Organization for Research and Treatment of Cancer Brain Tumor Group. Clin Cancer Res 16:1597–1604CrossRefGoogle Scholar
  29. 29.
    Sanson M, Marie Y, Paris S et al (2009) Isocitrate dehydrogenase 1 codon 132 mutation is an important prognostic biomarker in gliomas. J Clin Oncol 27:4150–4154CrossRefGoogle Scholar
  30. 30.
    Louis DN, Perry A, Reifenberger G et al (2016) The 2016 World Health Organization classification of tumors of the central nervous system: a summary. Acta Neuropathol 131:803–820CrossRefGoogle Scholar
  31. 31.
    Zou P, Xu H, Chen P et al (2013) IDH1/IDH2 Mutations define the prognosis and molecular profiles of patients with gliomas: a meta-analysis. PLoS ONE 8:e68782CrossRefGoogle Scholar
  32. 32.
    Molenaar RJ, Verbaan D, Lamba S et al (2014) The combination of IDH1 mutations and MGMT methylation status predicts survival in glioblastoma better than either IDH1 or MGMT alone. Neuro Oncol 16:1263–1273CrossRefGoogle Scholar
  33. 33.
    Millward CP, Brodbelt AR, Haylock B et al (2016) The impact of MGMT methylation and IDH-1 mutation on long-term outcome for glioblastoma treated with chemoradiotherapy. Acta Neurochir 158:1943–1953CrossRefGoogle Scholar
  34. 34.
    Zhang J, Yang JH, Quan J et al (2016) Identification of MGMT promoter methylation sites correlating with gene expression and IDH1 mutation in gliomas. Tumour Biol 37:13571–13579CrossRefGoogle Scholar
  35. 35.
    Felsberg J, Wolter M, Seul H et al (2010) Rapid and sensitive assessment of the IDH1 and IDH2 mutation status in cerebral gliomas based on DNA pyrosequencing. Acta Neuropathol 119:501–507CrossRefGoogle Scholar
  36. 36.
    Felsberg J, Rapp M, Loeser S et al (2009) Prognostic significance of molecular markers and extent of resection in primary glioblastoma patients. Clin Cancer Res 15:6683–6693CrossRefGoogle Scholar
  37. 37.
    Weller M, Felsberg J, Hartmann C et al (2009) Molecular predictors of progression-free and overall survival in patients with newly diagnosed glioblastoma: a prospective translational study of the German Glioma Network. J Clin Oncol 27:5743–5750CrossRefGoogle Scholar
  38. 38.
    Minniti G, Salvati M, Arcella A et al (2011) Correlation between O6-methylguanine-DNA methyltransferase and survival in elderly patients with glioblastoma treated with radiotherapy plus concomitant and adjuvant temozolomide. J Neurooncol 102:311–316CrossRefGoogle Scholar
  39. 39.
    Rand K, Qu W, Ho T et al (2002) Conversion-specific detection of DNA methylation using real-time polymerase chain reaction (ConLight-MSP) to avoid false positives. Methods 27:114–120CrossRefGoogle Scholar
  40. 40.
    Barault L, Amatu A, Bleeker FE et al (2015) Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer. Ann Oncol 26:1994–1999CrossRefGoogle Scholar
  41. 41.
    Kitange GJ, Carlson BL, Mladek AC et al (2009) Evaluation of MGMT promoter methylation status and correlation with temozolomide response in orthotopic glioblastoma xenograft model. J Neurooncol 92:23–31CrossRefGoogle Scholar
  42. 42.
    Parkinson JF, Wheeler HR, Clarkson A et al (2008) Variation of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation in serial samples in glioblastoma. J Neurooncol 87:71–78CrossRefGoogle Scholar
  43. 43.
    Wang S, Song C, Zha Y et al (2016) The prognostic value of MGMT promoter status by pyrosequencing assay for glioblastoma patients’ survival: a meta-analysis. World J Surg Oncol 14:261CrossRefGoogle Scholar
  44. 44.
    Kishida Y, Natsume A, Toda H et al (2012) Correlation between quantified promoter methylation and enzymatic activity of O 6-methylguanine-DNA methyltransferase in glioblastomas. Tumor Biol 33:373–381CrossRefGoogle Scholar
  45. 45.
    Quillien V, Lavenu A, Sanson M et al (2014) Outcome-based determination of optimal pyrosequencing assay for MGMT methylation detection in glioblastoma patients. J Neurooncol 116:487–496CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Elisa De Carlo
    • 1
    • 2
  • Lorenzo Gerratana
    • 1
    • 3
  • Giovanna De Maglio
    • 4
  • Vanessa Buoro
    • 1
    • 3
  • Francesco Cortiula
    • 1
    • 3
  • Lorena Gurrieri
    • 5
  • Miriam Isola
    • 3
  • Gianpiero Fasola
    • 1
  • Fabio Puglisi
    • 2
    • 3
  • Stefano Pizzolitto
    • 4
  • Simona Rizzato
    • 1
  1. 1.Department of OncologyUniversity Hospital of UdineUdineItaly
  2. 2.Department of Clinical OncologyIRCCS CRO Aviano National Cancer InstituteAvianoItaly
  3. 3.Department of Medicine (DAME)The University of UdineUdineItaly
  4. 4.Department of PathologyUniversity Hospital of UdineUdineItaly
  5. 5.Department of OncologyASUITS University HospitalTriesteItaly

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