Skip to main content
SpringerLink
Log in

Primer extension based quantitative polymerase chain reaction reveals consistent differences in the methylation status of the MGMT promoter in diffusely infiltrating gliomas (WHO grade II–IV) of adults

  • Clinical Study – Patient Study
  • Published:
Journal of Neuro-Oncology Aims and scope Submit manuscript

Abstract

Diffusely infiltrating gliomas (WHO grade II–IV) are the most common primary brain tumours in adults. These tumours are not amenable to cure by surgery alone, so suitable biomarkers for adjuvant modalities are required to guide therapeutic decision-making. Epigenetic silencing of the O6-methylguanine-DNA methyltransferase (MGMT) gene by promoter methylation has been associated with longer survival of patients with high-grade gliomas who receive alkylating chemotherapy; and molecular testing for the methylation status of the MGMT promoter sequence is regarded as among the most relevant of such markers. We have developed a primer extension-based assay adapted to formalin-fixed paraffin-embedded tissues that enables quantitative assessment of the methylation status of the MGMT promoter. The assay is very sensitive, highly reproducible, and provides valid test results in nearly 100% of cases. Our results indicate that oligodendrogliomas, empirically known to have a relatively favourable prognosis, are also the most homogeneous entities in terms of MGMT promoter methylation. Conversely, astrocytomas, which are more prone to spontaneous progression to higher grade malignancy, are significantly more heterogeneous. In addition, we show that the degree of promoter methylation correlates with the prevalence of loss of heterozygosity on chromosome arm 1p in the oligodendroglioma group, but not the astrocytoma group. Our results may have potentially important implications for clinical molecular diagnosis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

FFPE:

Formalin-fixed paraffin-embedded

LOH:

Loss of heterozygosity

MGMT:

O6-methylguanine-DNA methyltransferase

MSP:

Methylation-specific polymerase chain reaction

TMZ:

Temozolomide

References

  1. Louis D, Ohgaki H, Wiestler O, Cavenee W, Burger P, Jouvet A, Scheithauer B, Kleihues P (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114:97–109

    Article  PubMed  Google Scholar 

  2. van den Bent MJ, Hegi ME, Stupp R (2006) Recent developments in the use of chemotherapy in brain tumours. Eur J Cancer 42:582–588

    Article  PubMed  Google Scholar 

  3. Reifenberger G, Louis DN (2003) Oligodendroglioma: toward molecular definitions in diagnostic neuro-oncology. J Neuropathol Exp Neurol 62:111–126

    PubMed  CAS  Google Scholar 

  4. Smith JS, Perry A, Borell TJ, Lee H, O’Fallon J, Hosek S, Kimmel D, Yates A, Burger P, Scheithauer B, Jenkins R (2000) Alterations of chromosome arms 1p and 19q as predictors of survival in oligodendrogliomas, astrocytomas and mixed oligoastrocytomas. J Clin Oncol 18:636–645

    PubMed  CAS  Google Scholar 

  5. Thiessen B, Maguire J, McNeil K, Huntsman D, Martin MA, Horsman D (2003) Loss of heterozygosity for loci on chromosome arms 1p and 19q in oligodendroglioma tumours: relationship to outcome and chemosensitivity. J Neurooncol 64:271–278

    Article  PubMed  Google Scholar 

  6. Kujas M, Lejeune J, Benouaich-Amiel A, Crinière E, Laigle-Donadey F, Marie Y, Mokhtari K, Polivka M, Bernier M, Chretien F, Couvelard A, Capelle L, Duffau H, Cornu P, Broët P, Thillet J, Carpentier A, Sanson M, Hoang-Xuan K, Delattre J (2005) Chromosome 1p loss: a favorable prognostic factor in low-grade gliomas. Ann Neurol 58:322–326

    Article  PubMed  CAS  Google Scholar 

  7. Jenkins R, Blair H, Ballman KV, Giannini C, Arusell R, Law M, Flynn H, Passe S, Felten S, Brown P, Shaw E, Buckner J (2006) A t(1;19)(q10;p10) translocation mediates the combined deletions of 1p and 19q and predicts better prognosis of patients with oligodendroglioma. Cancer Res 66:9852–9861

    Article  PubMed  CAS  Google Scholar 

  8. Griffin C, Burger P, Morsberger L, Yonescu R, Swierczyknski S, Weingart JD, Murphy K (2006) Identification of der (1;19)(q10;p10) in five oligodendrogliomas suggests a mechanism of concurrent 1p and 19q loss. J Neuropathol Exp Neurol 65:988–994

    Article  PubMed  Google Scholar 

  9. Hoang-Xuan K, Capelle L, Kujas M, Taillibert S, Duffau H, Lejeune J, Polivka M, Crinière E, Marie Y, Mokhtari K, Carpentier A, Laigle F, Simon J, Cornu P, Broët P, Sanson M, Delattre J (2004) Temozolomide as initial treatment for adults with low-grade oligodendrogliomas or oligoastrocytomas and correlation with chromosome 1p deletions. J Clin Oncol 22:3133–3138

    Article  PubMed  CAS  Google Scholar 

  10. Mariani L, Deiana G, Vassella E, Fathi AR, Murtin C, Arnold M, Vajtai I, Weis J, Siegenthaler P, Schobesberger M, Reinert M (2006) Loss of heterozygosity 1p36 and 19q13 is a prognostic factor for overall survival in patients with diffuse WHO grade 2 gliomas treated without chemotherapy. J Clin Oncol 24:4758–4763

    Article  PubMed  CAS  Google Scholar 

  11. Hegi ME, Murat A, Lambiv WL, Stupp R (2006) Brain tumors: molecular biology and targeted therapies. Ann Oncol 17:191–197

    Article  Google Scholar 

  12. Esteller M, Garcia-Foncillas J, Andion E, Goodman SN, Hidalgo OF, Vanaclocha V, Baylin SB, Herman JG (2000) Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 343:1350–1354

    Article  PubMed  CAS  Google Scholar 

  13. Hegi ME, Diserens AC, Godard S, Dietrich PY, Regli L, Ostermann S, Otten P, Van Melle G, de Tribolet N, Stupp R (2004) Clinical trial substantiates the predictive value of O-6-methylguanine-DNA methyltransferase promoter methylation in glioblastoma patients treated with temozolomide. Clin Cancer Res 10:1871–1874

    Article  PubMed  CAS  Google Scholar 

  14. Hegi ME, Diserens A-C, Gorlia T, Hamou M, de Tribolet N, Weller M, Kros J, Hainfellner J, Mason W, Mariani L, Bromberg J, Hau P, Mirimanoff R, Cairncross J, Janzer RRS (2005) MGMT gene silencing and benefit from Temozolomide in glioblastoma. N Engl J Med 352:997–1003

    Article  PubMed  CAS  Google Scholar 

  15. Martinez R, Schackert G, Yaya-Tur R, Rojas-Marcos I, Herman JG, Esteller M (2007) Frequent hypermethylation of the DNA repair gene MGMT in long-term survivors of glioblastoma multiforme. J Neurooncol 83:91–93

    Article  PubMed  CAS  Google Scholar 

  16. Stupp R, Hegi ME, Gilbert MR, Chakravarti A (2007) Chemoradiotherapy in malignant glioma: standard of care and future directions. J Clin Oncol 25:4127–4136

    Article  PubMed  CAS  Google Scholar 

  17. Laird PW (2003) The power and the promise of DNA methylation markers. Nat Rev Cancer 253:253–266

    Article  Google Scholar 

  18. Karayan-Tapon L, Quillien V, Guihot J, Wager M, Fromont G, Saikali S, Etcheverry A, Hamlat A, Loussouarn D, Champion L, Campone M, Vallette F-M, Gratas-Rabbia-Re C (2010) Prognostic value of O6-methylguanine-DNA methyltransferase status in glioblastoma patients, assessed by five different methods. J Neurooncol 97:311–322

    Article  PubMed  CAS  Google Scholar 

  19. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K, Hau P, Brandes AA, Gijtenbeek J, Marosi C, Vecht CJ, Mokhtari K, Wesseling P, Villa S, Eisenhauer E, Gorlia T, Weller M, Lacombe D, Cairncross JG, Mirimanoff RO (2009) 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 10:459–466

    Article  PubMed  CAS  Google Scholar 

  20. Cankovic M, Mikkelsen T, Rosenblum ML, Zarbo RJ (2007) A simplified laboratory validated assay for MGMT promoter hypermethylation analysis of glioma specimens from formalin-fixed paraffin-embedded tissue. Lab Invest 87:392–397

    PubMed  CAS  Google Scholar 

  21. Vlassenbroeck I, Califice S, Diserens AC, Migliavacca E, Straub J, Di Stefano I, Moreau F, Hamou MF, Renard I, Delorenzi M, Flamion B, DiGuiseppi J, Bierau K, Hegi ME (2008) Validation of real-time methylation-specific PCR to determine O6-methylguanine-DNA methyltransferase gene promoter methylation in glioma. J Mol Diagn 10:332–337

    Article  PubMed  CAS  Google Scholar 

  22. Nancarrow D, HY H, Stark M, Whiteman D, NK H (2007) SiDCoN: a tool to aid scoring of DNA copy number changes in SNP chip data. PLoS One 2:e1093

    Article  PubMed  Google Scholar 

  23. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93:9821–9826

    Article  PubMed  CAS  Google Scholar 

  24. Mikeska T, Bock C, El-Maarri O, Hubner A, Ehrentraut D, Schramm J, Felsberg J, Kahl P, Buttner R, Pietsch T, Waha A (2007) Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis. J Mol Diagn 9:368–381

    Article  PubMed  CAS  Google Scholar 

  25. Hattermann K, Mehdorn HM, Mentlein R, Schultka S, Held-Feindt J (2008) A methylation-specific and SYBR-green-based quantitative polymerase chain reaction technique for O6-methylguanine DNA methyltransferase promoter methylation analysis. Anal Biochem 377:62–71

    Article  PubMed  CAS  Google Scholar 

  26. Gonzalgo ML, Jones PA (2002) Quantitative methylation analysis using methylation-sensitive single-nucleotide primer extension (Ms-SNuPE). Methods 27:128–133

    Article  PubMed  CAS  Google Scholar 

  27. Jeuken JW, Cornelissen SJ, Vriezen M, Dekkers MM, Errami A, Sijben A, Boots-Sprenger SH, Wesseling P (2007) MS-MLPA: an attractive alternative laboratory assay for robust, reliable, and semiquantitative detection of MGMT promoter hypermethylation in gliomas. Lab Invest 87:1055–1065

    Article  PubMed  CAS  Google Scholar 

  28. Mollemann M, Wolter M, Felsberg J, Collins VP, Reifenberger G (2005) Frequent promoter hypermethylation and low expression of the MGMT gene in oligodendroglial tumors. Int J Cancer 113:379–385

    Article  PubMed  Google Scholar 

  29. Christensen BC, Houseman EA, Marsit CJ, Zheng S, Wrensch MR, Wiemels JL, Nelson HH, Karagas MR, Padbury JF, Bueno R, Sugarbaker DJ, Yeh RF, Wiencke JK, Kelsey KT (2009) Aging and environmental exposures alter tissue-specific DNA methylation dependent upon CpG island context. PLoS Genet 5:e1000602

    Article  PubMed  Google Scholar 

  30. Fraga MF, Agrelo R, Esteller M (2007) Cross-talk between aging and cancer: the epigenetic language. Ann NY Acad Sci 1100:60–74

    Article  PubMed  CAS  Google Scholar 

  31. Parrella P, la Torre A, Copetti M, Valori VM, Barbano R, Notarangelo A, Bisceglia M, Gallo AP, Balsamo T, Poeta ML, Carella M, Catapano D, Parisi S, Dallapiccola B, Maiello E, D’Angelo V, Fazio VM (2009) High specificity of quantitative methylation-specific PCR analysis for MGMT promoter hypermethylation detection in gliomas. J Biomed Biotechnol 2009:531692

    Article  PubMed  Google Scholar 

  32. Preusser M (2009) MGMT analysis at DNA, RNA and protein levels in glioblastoma tissue. Histol Histopathol 24:511–518

    PubMed  CAS  Google Scholar 

  33. Weller M, Stupp R, Reifenberger G, Brandes AA, van den Bent MJ, Wick W, Hegi ME (2010) MGMT promoter methylation in malignant gliomas: ready for personalized medicine? Nat Rev Neurol 6:39–51

    Article  PubMed  CAS  Google Scholar 

  34. Dunn J, Baborie A, Alam F, Joyce K, Moxham M, Sibson R, Crooks D, Husband D, Shenoy A, Brodbelt A, Wong H, Liloglou T, Haylock B, Walker C (2009) Extent of MGMT promoter methylation correlates with outcome in glioblastomas given temozolomide and radiotherapy. Br J Cancer 101:124–131

    Article  PubMed  CAS  Google Scholar 

  35. Juillerat-Jeanneret L, Bernasconi CC, Bricod C, Gros S, Trepey S, Benhattar J, Janzer RC (2008) Heterogeneity of human glioblastoma: glutathione-S-transferase and methylguanine-methyltransferase. Cancer Invest 26:597–609

    Article  PubMed  CAS  Google Scholar 

  36. Parkinson JF, Wheeler HR, Clarkson A, McKenzie CA, Biggs MT, Little NS, Cook RJ, Messina M, Robinson BG, McDonald KL (2008) Variation of O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation in serial samples in glioblastoma. J Neurooncol 87:71–78

    Article  PubMed  CAS  Google Scholar 

  37. Grasbon-Frodl EM, Kreth FW, Ruiter M, Schnell O, Bise K, Felsberg J, Reifenberger G, Tonn JC, Kretzschmar HA (2007) Intratumoral homogeneity of MGMT promoter hypermethylation as demonstrated in serial stereotactic specimens from anaplastic astrocytomas and glioblastomas. Int J Cancer 121:2458–2464

    Article  PubMed  CAS  Google Scholar 

  38. Ochsenbein AF, Schubert AD, Vassella E, Mariani L (2010) Quantitative analysis of O(6)-methylguanine DNA methyltransferase (MGMT) promoter methylation in patients with low-grade gliomas. J Neurooncol [Epub ahead of print]

  39. van den Bent MJ, Dubbink HJ, Sanson M, van der Lee-Haarloo CR, Hegi M, Jeuken JW, Ibdaih A, Brandes AA, Taphoorn MJ, Frenay M, Lacombe D, Gorlia T, Dinjens WN, Kros JM (2009) MGMT promoter methylation is prognostic but not predictive for outcome to adjuvant PCV chemotherapy in anaplastic oligodendroglial tumors: a report from EORTC Brain Tumor Group Study 26951. J Clin Oncol 27:5881–5886

    Article  PubMed  Google Scholar 

  40. Wick W, Hartmann C, Engel C, Stoffels M, Felsberg J, Stockhammer F, Sabel MC, Koeppen S, Ketter R, Meyermann R, Rapp M, Meisner C, Kortmann RD, Pietsch T, Wiestler OD, Ernemann U, Bamberg M, Reifenberger G, von Deimling A, Weller M (2009) NOA-04 randomized phase III trial of sequential radiochemotherapy of anaplastic glioma with procarbazine, lomustine, and vincristine or temozolomide. J Clin Oncol 27:5874–5880

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by a grant from the Swiss National Science Foundation and the Bernese Cancer League.

Author information

Authors and Affiliations

  1. Institute of Pathology, University of Bern, Murtenstrasse 31, 3010, Bern, Switzerland

    Erik Vassella, Istvan Vajtai, Nora Bandi, Marlene Arnold & Verena Kocher

  2. Department of Neurosurgery, University Hospital of Basel, Basel, 4031, Switzerland

    Luigi Mariani

Authors
  1. Erik Vassella
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Istvan Vajtai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nora Bandi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marlene Arnold
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Verena Kocher
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Luigi Mariani
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Erik Vassella.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vassella, E., Vajtai, I., Bandi, N. et al. Primer extension based quantitative polymerase chain reaction reveals consistent differences in the methylation status of the MGMT promoter in diffusely infiltrating gliomas (WHO grade II–IV) of adults. J Neurooncol 104, 293–303 (2011). https://doi.org/10.1007/s11060-010-0490-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11060-010-0490-4

Keywords

Search

Navigation