Brain Tumor Pathology

, Volume 28, Issue 1, pp 25–31 | Cite as

Quantitative digital assessment of MGMT immunohistochemical expression in glioblastoma tissue

  • Yukie Araki
  • Masahiro Mizoguchi
  • Koji Yoshimoto
  • Tadahisa Shono
  • Toshiyuki Amano
  • Akira Nakamizo
  • Satoshi O. Suzuki
  • Toru Iwaki
  • Tomio Sasaki
Original Article


Recent reports have suggested an important clinical role for hypermethylation of the O6-methylguanine-DNA-methyltransferase (MGMT) promoter in patients with glioblastomas. Whether MGMT protein expression is correlated with promoter hypermethylation and patient outcomes, however, has not been elucidated. Here we describe a quantitative digital method for assessment of MGMT-specific immunostaining, and analyze the relationship between expression levels and methylation status of the MGMT promoter. We investigated 46 tumors from patients who received a diagnosis of glioblastoma or gliosarcoma. Immunohistochemistry with anti-MGMT antibody and methylation-specific PCR using bisulfite-modified tumor DNA were performed. The digital assessment method used image-analysis software to determine a digital MGMT staining index, and the results were compared with those obtained via conventional visual assessments. The digital staining index clearly correlated with the methylation status of MGMT promoter. In addition, the index correlated with our observational results when nuclear and cytoplasmic staining were assessed in three different fields. Our digital assessment method enabled us to assess uncertain immunopositive samples objectively and quantitatively, which is an important consideration when examining heterogeneous cellular staining. We expect that this method will be useful for assessment of heterogeneous staining with any antibodies.


O6-methylguanine methyltransferase (MGMT) Glioblastoma Immunohistochemistry Quantitative assessment 


  1. 1.
    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 343:1350–1354CrossRefPubMedGoogle Scholar
  2. 2.
    Stupp R, Mason WP, van den Bent MJ et al (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996CrossRefPubMedGoogle Scholar
  3. 3.
    Liu L, Gerson SL (2006) Targeted modulation of MGMT: clinical implications. Clin Cancer Res 12:328–331CrossRefPubMedGoogle Scholar
  4. 4.
    Brell M, Tortosa A, Verger E et al (2005) Prognostic significance of O6-methylguanine-DNA methyltransferase determined by promoter hypermethylation and immunohistochemical expression in anaplastic gliomas. Clin Cancer Res 11:5167–5174CrossRefPubMedGoogle Scholar
  5. 5.
    Cankovic M, Mikkelsen T, Rosenblum ML et al (2007) A simplified laboratory validated assay for MGMT promoter hypermethylation analysis of glioma specimens from formalin-fixed paraffin-embedded tissue. Lab Invest 87:392–397PubMedGoogle Scholar
  6. 6.
    Cao VT, Jung TY, Jung S et al (2009) The correlation and prognostic significance of MGMT promoter methylation and MGMT protein in glioblastomas. Neurosurgery 65:866–875CrossRefPubMedGoogle Scholar
  7. 7.
    Karayan-Tapon L, Quillien V, Guilhot J et al (2010) Prognostic value of O(6)-methylguanine-DNA methyltransferase status in glioblastoma patients, assessed by five different methods. J Neurooncol 97:311–322CrossRefPubMedGoogle Scholar
  8. 8.
    Parkinson JF, Wheeler HR, Clarkson A et al (2008) Variation of O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation in serial samples in glioblastoma. J Neurooncol 87:71–78CrossRefPubMedGoogle Scholar
  9. 9.
    Sonoda Y, Yokosawa M, Saito R et al (2010) O(6)-Methylguanine DNA methyltransferase determined by promoter hypermethylation and immunohistochemical expression is correlated with progression-free survival in patients with glioblastoma. Int J Clin Oncol 15:352–358CrossRefPubMedGoogle Scholar
  10. 10.
    Maxwell JA, Johnson SP, Quinn JA et al (2006) Quantitative analysis of O6-alkylguanine-DNA alkyltransferase in malignant glioma. Mol Cancer Ther 5:2531–2539CrossRefPubMedGoogle Scholar
  11. 11.
    Metellus P, Coulibaly B, Nanni I et al (2009) Prognostic impact of O6-methylguanine-DNA methyltransferase silencing in patients with recurrent glioblastoma multiforme who undergo surgery and carmustine wafer implantation: a prospective patient cohort. Cancer 115:4783–4794CrossRefPubMedGoogle Scholar
  12. 12.
    Sasai K, Nodashira M, Nishihara H et al (2008) Careful exclusion of non-neoplastic brain components is required for an appropriate evaluation of O6-methylguanine-DNA methyltransferase status in glioma: relationship between immunohistochemistry and methylation analysis. Am J Surg Pathol 32:1220–1227CrossRefPubMedGoogle Scholar
  13. 13.
    Preusser M, Janzer RC, Felsberg J et al (2008) Anti-O6-methylguanine-methyltransferase (MGMT) immunohistochemistry in glioblastoma multiforme: observer variability and lack of association with patient survival impede its use as clinical biomarker. Brain Pathol 18:520–532PubMedGoogle Scholar
  14. 14.
    Anda T, Shabani HK, Tsunoda K et al (2003) Relationship between expression of O6-methylguanine-DNA methyltransferase, glutathione-S-transferase pi in glioblastoma and the survival of the patients treated with nimustine hydrochloride: an immunohistochemical analysis. Neurol Res 25:241–248CrossRefPubMedGoogle Scholar
  15. 15.
    Preusser M (2009) MGMT analysis at DNA, RNA and protein levels in glioblastoma tissue. Histol Histopathol 24:511–518PubMedGoogle Scholar
  16. 16.
    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 15:6683–6693CrossRefPubMedGoogle Scholar
  17. 17.
    Grasbon-Frodl EM, Kreth FW, Ruiter M et al (2007) Intratumoral homogeneity of MGMT promoter hypermethylation as demonstrated in serial stereotactic specimens from anaplastic astrocytomas and glioblastomas. Int J Cancer 121:2458–2464CrossRefPubMedGoogle Scholar
  18. 18.
    Mollemann M, Wolter M, Felsberg J et al (2005) Frequent promoter hypermethylation and low expression of the MGMT gene in oligodendroglial tumors. Int J Cancer 113:379–385CrossRefPubMedGoogle Scholar
  19. 19.
    Nakagawa T, Ido K, Sakuma T et al (2009) Prognostic significance of the immunohistochemical expression of O(6)-methylguanine-DNA methyltransferase, P-glycoprotein, and multidrug resistance protein-1 in glioblastomas. Neuropathology 29:379–388CrossRefPubMedGoogle Scholar
  20. 20.
    Nakasu S, Fukami T, Baba K et al (2004) Immunohistochemical study for O6-methylguanine-DNA methyltransferase in the non-neoplastic and neoplastic components of gliomas. J Neurooncol 70:333–340CrossRefPubMedGoogle Scholar
  21. 21.
    Sonoda Y, Kumabe T, Watanabe M et al (2009) Long-term survivors of glioblastoma: clinical features and molecular analysis. Acta Neurochir (Wien) 151:1349–1358CrossRefGoogle Scholar
  22. 22.
    Yachi K, Watanabe T, Ohta T et al (2008) Relevance of MSP assay for the detection of MGMT promoter hypermethylation in glioblastomas. Int J Oncol 33:469–475PubMedGoogle Scholar
  23. 23.
    Cloughesy TF, Yoshimoto K, Nghiemphu P et al (2008) Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma. PLoS Med 5:e8CrossRefPubMedGoogle Scholar
  24. 24.
    Mellinghoff IK, Wang MY, Vivanco I et al (2005) Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors. N Engl J Med 353:2012–2024CrossRefPubMedGoogle Scholar
  25. 25.
    Kleihues P, Burger PC, Aldape KD, Brat DJ et al (2007) Glioblastoma. In: Louis DN, Ohgaki H, Wiestler OD et al (eds) WHO classification of tumours of the central nervous system, 4th edn. IARC, Lyon, pp 33–49Google Scholar
  26. 26.
    Eoli M, Pollo B, Bianchessi F et al (2006) Molecular markers of gliomas: a clinical approach. Neurol Res 28:538–541CrossRefPubMedGoogle Scholar
  27. 27.
    Stupp R, Hegi ME, Mason WP et al (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 10:459–466CrossRefPubMedGoogle Scholar
  28. 28.
    Hegi ME, Diserens AC, Gorlia T et al (2005) MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352(10):997–1003CrossRefPubMedGoogle Scholar
  29. 29.
    Stupp R, Hegi ME, Mark R et al (2007) Chemoradiotherapy in malignant glioma: standard of care and future directions. J Clin Oncol 25:4127–4136CrossRefPubMedGoogle Scholar
  30. 30.
    Gerson SL (2002) Clinical relevance of MGMT in the treatment of cancer. J Clin Oncol 20:2388–2399CrossRefPubMedGoogle Scholar
  31. 31.
    Srivenugopal KS, Yuan XH, Friedman HS et al (1996) Ubiquitination-dependent proteolysis of O6-methylguanine-DNA methyltransferase in human and murine tumor cells following inactivation with O6-benzylguanine or 1,3-bis(2-chloroethyl)-1-nitrosourea. Biochemistry 35:1328–1334CrossRefPubMedGoogle Scholar
  32. 32.
    Zaidi NH, Liu L, Gerson SL (1996) Quantitative immunohistochemical estimates of O6-alkylguanine-DNA alkyltransferase expression in normal and malignant human colon. Clin Cancer Res 2:577–584PubMedGoogle Scholar

Copyright information

© The Japan Society of Brain Tumor Pathology 2011

Authors and Affiliations

  • Yukie Araki
    • 1
  • Masahiro Mizoguchi
    • 1
  • Koji Yoshimoto
    • 1
  • Tadahisa Shono
    • 1
  • Toshiyuki Amano
    • 1
  • Akira Nakamizo
    • 1
  • Satoshi O. Suzuki
    • 2
  • Toru Iwaki
    • 2
  • Tomio Sasaki
    • 1
  1. 1.Department of Neurosurgery, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan
  2. 2.Department of Neuropathology, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan

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