Journal of Neuro-Oncology

, Volume 103, Issue 1, pp 59–69 | Cite as

Expression of O6-methylguanine-DNA methyltransferase in childhood medulloblastoma

  • Denis Faoro
  • André O. von Bueren
  • Tarek Shalaby
  • Davide Sciuscio
  • Marie-Louise Hürlimann
  • Lucia Arnold
  • Nicolas U. Gerber
  • Johannes Haybaeck
  • Michel Mittelbronn
  • Stefan Rutkowski
  • Monika Hegi
  • Michael A. Grotzer
Laboratory Investigation - Human/Animal Tissue


Medulloblastomas (MB) are the most common malignant brain tumors in childhood. Alkylator-based drugs are effective agents in the treatment of patients with MB. In several tumors, including malignant glioma, elevated O6-methylguanine-DNA methyltransferase (MGMT) expression levels or lack of MGMT promoter methylation have been found to be associated with resistance to alkylating chemotherapeutic agents such as temozolomide (TMZ). In this study, we examined the MGMT status of MB and central nervous system primitive neuroectodermal tumor (PNET) cells and two large sets of primary MB. In seven MB/PNET cell lines investigated, MGMT promoter methylation was detected only in D425 human MB cells as assayed by the qualitative methylation-specific PCR and the more quantitative pyrosequencing assay. In D425 human MB cells, MGMT mRNA and protein expression was clearly lower when compared with the MGMT expression in the other MB/PNET cell lines. In MB/PNET cells, sensitivity towards TMZ and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) correlated with MGMT methylation and MGMT mRNA expression. Pyrosequencing in 67 primary MB samples revealed a mean percentage of MGMT methylation of 3.7–92% (mean: 13.25%, median: 10.67%). Percentage of MGMT methylation and MGMT mRNA expression as determined by quantitative RT-PCR correlated inversely (n = 46; Pearson correlation r 2 = 0.14, P = 0.01). We then analyzed MGMT mRNA expression in a second set of 47 formalin-fixed paraffin-embedded primary MB samples from clinically well-documented patients treated within the prospective randomized multicenter trial HIT’91. No association was found between MGMT mRNA expression and progression-free or overall survival. Therefore, it is not currently recommended to use MGMT mRNA expression analysis to determine who should receive alkylating agents and who should not.


Childhood brain tumors Medulloblastoma MGMT Methylation 


  1. 1.
    Gurney JG, Smith MA, Bunin GR (2000) CNS and miscellaneous intracranial and intraspinal neoplasms. In: SEER pediatric monograph. National Cancer Institute, pp 51–63.
  2. 2.
    Packer RJ (1999) Alternative treatments for childhood brain tumors. Childs Nerv Syst 15:789–794PubMedCrossRefGoogle Scholar
  3. 3.
    Douglas JG, Barker JL, Ellenbogen RG, Geyer JR (2004) Concurrent chemotherapy and reduced-dose cranial spinal irradiation followed by conformal posterior fossa tumor bed boost for average-risk medulloblastoma: efficacy and patterns of failure. Int J Radiat Oncol Biol Phys 58(4):1161–1164PubMedCrossRefGoogle Scholar
  4. 4.
    Gerson SL (2004) MGMT: its role in cancer aetiology and cancer therapeutics. Nat Rev Cancer 4(4):296–307PubMedCrossRefGoogle Scholar
  5. 5.
    Tano K, Shiota S, Collier J, Foote RS, Mitra S (1990) Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine. Proc Natl Acad Sci USA 87(2):686–690PubMedCrossRefGoogle Scholar
  6. 6.
    Gerson SL (2002) Clinical relevance of MGMT in the treatment of cancer. J Clin Oncol 20(9):2388–2399PubMedCrossRefGoogle Scholar
  7. 7.
    Pegg AE (1990) Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents. Cancer Res 50(19):6119–6129PubMedGoogle Scholar
  8. 8.
    Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG (1999) Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 59(4):793–797PubMedGoogle Scholar
  9. 9.
    Pegg AE, Dolan ME, Moschel RC (1995) Structure, function, and inhibition of O6-alkylguanine-DNA alkyltransferase. Prog Nucleic Acid Res Mol Biol 51:167–223PubMedCrossRefGoogle Scholar
  10. 10.
    Gonzaga PE, Brent TP (1989) Affinity purification and characterization of human O6-alkylguanine-DNA alkyltransferase complexed with BCNU-treated, synthetic oligonucleotide. Nucleic Acids Res 17(16):6581–6590PubMedCrossRefGoogle Scholar
  11. 11.
    Coulondre C, Miller JH (1977) Genetic studies of the lac repressor. IV. Mutagenic specificity in the lacI gene of Escherichia coli. J Mol Biol 117(3):577–606PubMedCrossRefGoogle Scholar
  12. 12.
    Aquilina G, Hess P, Branch P, MacGeoch C, Casciano I, Karran P, Bignami M (1994) A mismatch recognition defect in colon carcinoma confers DNA microsatellite instability and a mutator phenotype. Proc Natl Acad Sci USA 91(19):8905–8909PubMedCrossRefGoogle Scholar
  13. 13.
    Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, Kros JM, Hainfellner JA, Mason W, Mariani L, Bromberg JE, Hau P, Mirimanoff RO, Caincross JG, Janzer RC, Stupp R (2005) MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352(10):997–1003PubMedCrossRefGoogle Scholar
  14. 14.
    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(6):1871–1874PubMedCrossRefGoogle Scholar
  15. 15.
    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(19):1350–1354PubMedCrossRefGoogle Scholar
  16. 16.
    Pollack IF, Hamilton RL, Sobol RW, Burnham J, Yates AJ, Holmes EJ, Zhou T, Finlay JL (2006) O6-methylguanine-DNA methyltransferase expression strongly correlates with outcome in childhood malignant gliomas: results from the CCG-945 cohort. J Clin Oncol 24(21):3431–3437PubMedCrossRefGoogle Scholar
  17. 17.
    He XM, Ostrowski LE, von Wronski MA, Friedman HS, Wikstrand CJ, Bigner SH, Rasheed A, Batra SK, Mitra S, Brent TP (1992) Expression of O6-methylguanine-DNA methyltransferase in six human medulloblastoma cell lines. Cancer Res 52(5):1144–1148PubMedGoogle Scholar
  18. 18.
    Hongeng S, Brent TP, Sanford RA, Li H, LE Kun, Heideman RL (1997) O6-Methylguanine-DNA methyltransferase protein levels in pediatric brain tumors. Clin Cancer Res 3(12 Pt 1):2459–2463PubMedGoogle Scholar
  19. 19.
    Rood BR, Zhang H, Cogen PH (2004) Intercellular heterogeneity of expression of the MGMT DNA repair gene in pediatric medulloblastoma. Neuro-oncology 6(3):200–207PubMedCrossRefGoogle Scholar
  20. 20.
    Neben K, Korshunov A, Benner A, Wrobel G, Hahn M, Kokocinski F, Golanov A, Joos S, Lichter P (2004) Microarray-based screening for molecular markers in medulloblastoma revealed STK15 as independent predictor for survival. Cancer Res 64(9):3103–3111PubMedCrossRefGoogle Scholar
  21. 21.
    Bobola MS, Silber JR, Ellenbogen RG, Geyer JR, Blank A, Goff RD (2005) O6-methylguanine-DNA methyltransferase, O6-benzylguanine, and resistance to clinical alkylators in pediatric primary brain tumor cell lines. Clin Cancer Res 11(7):2747–2755PubMedCrossRefGoogle Scholar
  22. 22.
    von Bueren AO, Shalaby T, Rajtarova J, Stearns D, Eberhart CG, Helson L, Arcaro A, Grotzer MA (2007) Anti-proliferative activity of the quassinoid NBT-272 in childhood medulloblastoma cells. BMC Cancer 7(1):19CrossRefGoogle Scholar
  23. 23.
    Bieche I, Nogues C, Paradis V, Olivi M, Bedossa P, Lidereau R, Vidaud M (2000) Quantitation of hTERT gene expression in sporadic breast tumors with a real-time reverse transcription-polymerase chain reaction assay. Clin Cancer Res 6(2):452–459PubMedGoogle Scholar
  24. 24.
    Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25(4):386–401PubMedCrossRefGoogle Scholar
  25. 25.
    Mengelle C, Sandres-Saune K, Pasquier C, Rostaing L, Mansuy JM, Marty M, Da Silva I, Attal M, Massip P, Izopet J (2003) Automated extraction and quantification of human cytomegalovirus DNA in whole blood by real-time PCR assay. J Clin Microbiol 41(8):3840–3845PubMedCrossRefGoogle Scholar
  26. 26.
    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(4):332–337PubMedCrossRefGoogle Scholar
  27. 27.
    Mikeska T, Bock C, El-Maarri O, Hübner A, Ehrentraut D, Schramm J, Felsberg J, Kahl P, Büttner R, Pietsch T, Waha A (2007) Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis. J Mol Diagn 9(3):368–381PubMedCrossRefGoogle Scholar
  28. 28.
    Rietschel P, Wolchok JD, Krown S, Gerst S, Jungbluth AA, Busam K, Smith K, Orlow I, Panageas K, Chapman PB (2008) Phase II study of extended-dose temozolomide in patients with melanoma. J Clin Oncol 26(14):2299–2304PubMedCrossRefGoogle Scholar
  29. 29.
    Ringquist S, Styche A, Rudert WA, Trucco M (2007) Pyrosequencing-based strategies for improved allele typing of human leukocyte antigen loci. Methods Mol Biol 373:115–134PubMedGoogle Scholar
  30. 30.
    Brakensiek K, Wingen LU, Langer F, Kreipe H, Lehmann U (2007) Quantitative high-resolution CpG island mapping with pyrosequencing reveals disease-specific methylation patterns of the CDKN2B gene in myelodysplastic syndrome and myeloid leukemia. Clin Chem 53(1):17–23PubMedCrossRefGoogle Scholar
  31. 31.
    Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, Zhang L, Score J, Seear R, Chase AJ, Grand FH, White H, Zoi C, Loukopoulos D, Terpos E, Vervessou EC, Schultheis B, Emig M, Ernst T, Lengfelder E, Hehlmann R, Hochhaus A, Oscier D, Silver RT, Reiter A, Cross NC (2005) Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 106(6):2162–2168PubMedCrossRefGoogle Scholar
  32. 32.
    Shaw RJ, Liloglou T, Rogers SN, Brown JS, Vaughan ED, Lowe D, Field JK, Risk JM (2006) Promoter methylation of P16, RARbeta, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing. Br J Cancer 94(4):561–568PubMedCrossRefGoogle Scholar
  33. 33.
    Liu L, Schwartz S, Davis BM, Gerson SL (2002) Chemotherapy-induced O(6)-benzylguanine-resistant alkyltransferase mutations in mismatch-deficient colon cancer. Cancer Res 62(11):3070–3076PubMedGoogle Scholar
  34. 34.
    Grotzer MA, Eggert A, Zuzak TJ, Janss AJ, Marwaha S, Wiewrodt BR, Ikegaki N, Brodeur CM, Phillips PC (2000) Resistance to TRAIL-induced apoptosis in primitive neuroectodermal brain tumor cells correlates with a loss of caspase-8 expression. Oncogene 19(40):4604–4610PubMedCrossRefGoogle Scholar
  35. 35.
    Cejka P, Stojic L, Mojas N, Russell AM, Heimimann K, Cannavo E, di Pietro M, Marra G, Jiricny J (2003) Methylation-induced G(2)/M arrest requires a full complement of the mismatch repair protein hMLH1. EMBO J 22(9):2245–2254PubMedCrossRefGoogle Scholar
  36. 36.
    Kortmann RD, Kuhl J, Timmermann B, Mittler U, Urban C, Budach V, Richter E, Willich N, Flentje M, Berthold F, Slavc I, Wolff J, Meisner C, Wiestler O, Sörensen N, Warmuth-Meth M, Bamberg M (2000) Postoperative neoadjuvant chemotherapy before radiotherapy as compared to immediate radiotherapy followed by maintenance chemotherapy in the treatment of medulloblastoma in childhood: results of the German prospective randomized trial HIT ‘91. Int J Radiat Oncol Biol Phys 46(2):269–279PubMedCrossRefGoogle Scholar
  37. 37.
    Rutkowski S, von Bueren A, von Hoff K, Hartmann W, Shalaby T, Deinlein F, Warmuth-Metz M, Soerensen N, Emser A, Bode U, Mittler U, Urban C, Benesch M, Kortmann RD, Kortmann RD, Schlegel PG, Kuehl J, Pietsch T, Grotzer M (2007) Prognostic relevance of clinical and biological risk factors in childhood medulloblastoma: results of patients treated in the prospective multicenter trial HIT’91. Clin Cancer Res 13(9):2651–2657PubMedCrossRefGoogle Scholar
  38. 38.
    Bobola MS, Tseng SH, Blank A, Berger MS, Silber JR (1996) Role of O6-methylguanine-DNA methyltransferase in resistance of human brain tumor cell lines to the clinically relevant methylating agents temozolomide and streptozotocin. Clin Cancer Res 2(4):735–741PubMedGoogle Scholar
  39. 39.
    Friedman HS, Keir S, Pegg AE, Houghton PJ, Colvin OM, Moschel RC, Bigner DD, Dolan ME (2002) O6-benzylguanine-mediated enhancement of chemotherapy. Mol Cancer Ther 1(11):943–948PubMedGoogle Scholar
  40. 40.
    Bacolod MD, Johnson SP, Pegg AE, Dolan ME, Moschel RC, Bullock NS, Fang Q, Colvin OM, Modrich P, Bigner DD, Friedman HS (2004) Brain tumor cell lines resistant to O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea chemotherapy have O6-alkylguanine-DNA alkyltransferase mutations. Mol Cancer Ther 3(9):1127–1135PubMedGoogle Scholar
  41. 41.
    Hegi ME, Liu L, Herman JG, Stupp R, Wick W, Weller M, Mehta MB, Gilbert MR (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(25):4189–4199PubMedCrossRefGoogle Scholar
  42. 42.
    Preusser M, Janzer CR, Felsber J, Reifenberger G, Hamou MF, Diserens AC, Stupp R, Gorlia T, Marosi C, Heinzl H, Hainfellner JA, Hegi M (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(4):520–532PubMedGoogle Scholar
  43. 43.
    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(18):9821–9826PubMedCrossRefGoogle Scholar
  44. 44.
    Shen L, Kondo Y, Rosner GL, Xiao L, Hernandez NS, Vilaythong J, Houlihan PS, Krouse RS, Prasad AR, Einspahr JG, Buckmeier J, Alberts DS, Hamilton SR, Issa JP (2005) MGMT promoter methylation and field defect in sporadic colorectal cancer. J Natl Cancer Inst 97(18):1330–1338PubMedCrossRefGoogle Scholar
  45. 45.
    Tost J, Gut IG (2007) DNA methylation analysis by pyrosequencing. Nat Protoc 2(9):2265–2275PubMedCrossRefGoogle Scholar
  46. 46.
    Lashford LS, Thiesse P, Jouvet A, Jaspan T, Couanet D, Griffiths PD, Doz F, Ironside J, Robson K, Hobson R, Dugan M, Pearson AD, Vassal G, Frappaz D (2002) Temozolomide in malignant gliomas of childhood: a United Kingdom Children’s Cancer Study Group and French Society for Pediatric Oncology Intergroup Study. J Clin Oncol 20(24):4684–4691PubMedCrossRefGoogle Scholar
  47. 47.
    Baruchel S, Diezi M, Hargrave D, Stempak D, Gammon J, Moghrabi A, Coppes MJ, Fernandez CV, Bouffet E (2006) Safety and pharmacokinetics of temozolomide using a dose-escalation, metronomic schedule in recurrent paediatric brain tumours. Eur J Cancer 42(14):2335–2342PubMedCrossRefGoogle Scholar
  48. 48.
    Kanzawa T, Germano IM, Komata T, Ito H, Kondo Y, Kondo S (2004) Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ 11(4):448–457PubMedCrossRefGoogle Scholar
  49. 49.
    Hermisson M, Klumpp A, Wick W, Wischhusen J, Nagel G, Roos W, Kaina B, Weller M (2006) O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells. J Neurochem 96(3):766–776PubMedCrossRefGoogle Scholar
  50. 50.
    Lewandowicz GM, Harding B, Harkness W, Hayward R, Thomas DG, Darling JL (2000) Chemosensitivity in childhood brain tumours in vitro: evidence of differential sensitivity to lomustine (CCNU) and vincristine. Eur J Cancer 36(15):1955–1964PubMedCrossRefGoogle Scholar
  51. 51.
    Wick A, Wick W, Hirrlinger J, Gerhardt E, Dringen R, Dichigans J, Weller M, Schulz JB (2004) Chemotherapy-induced cell death in primary cerebellar granule neurons but not in astrocytes: in vitro paradigm of differential neurotoxicity. J Neurochem 91(5):1067–1074PubMedCrossRefGoogle Scholar
  52. 52.
    Stojic L, Brun R, Jiricny J (2004) Mismatch repair and DNA damage signalling. DNA Repair 3(8–9):1091–1101PubMedCrossRefGoogle Scholar
  53. 53.
    Brock CS, Newlands ES, Wedge SR, Bower M, Evans H, Colquhoun I, Roddie M, Glaser M, Brampton MH, Rustin GJ (1998) Phase I trial of temozolomide using an extended continuous oral schedule. Cancer Res 58(19):4363–4367PubMedGoogle Scholar
  54. 54.
    Newlands ES, Blackledge GR, Slack JA, Rustin GJ, Smith DB, Stuart NS, Quarterman CP, Hoffman R, Stevens MF, Brampton MH (1992) Phase I trial of temozolomide (CCRG 81045: M&B 39831: NSC 362856). Br J Cancer 65(2):287–291PubMedCrossRefGoogle Scholar
  55. 55.
    Newlands ES, Stevens MF, Wedge SR, Wheelhouse RT, Brock C (1997) Temozolomide: a review of its discovery, chemical properties, pre-clinical development and clinical trials. Cancer Treat Rev 23(1):35–61PubMedCrossRefGoogle Scholar
  56. 56.
    Spiro TP, Liu L, Majka S, Haaga J, Willson JL, Gerson SL (2001) Temozolomide: the effect of once- and twice-a-day dosing on tumor tissue levels of the DNA repair protein O(6)-alkylguanine-DNA-alkyltransferase. Clin Cancer Res 7(8):2309–2317PubMedGoogle Scholar
  57. 57.
    Su YB, Sohn S, Krown SE, Livingston PO, Wolchok JD, Quinn C, Williams L, Foster T, Sepkowitz KA, chapman PB (2004) Selective CD4+ lymphopenia in melanoma patients treated with temozolomide: a toxicity with therapeutic implications. J Clin Oncol 22(4):610–616PubMedCrossRefGoogle Scholar
  58. 58.
    Ganiere V, Christen G, Bally F, Guillou L, Pica A, de Ribaupierre S, Stupp R (2006) Listeria brain abscess, Pneumocystis pneumonia and Kaposi’s sarcoma after temozolomide. Nat Clin Pract Oncol 3(6):339–343PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  • Denis Faoro
    • 1
  • André O. von Bueren
    • 2
  • Tarek Shalaby
    • 1
  • Davide Sciuscio
    • 3
  • Marie-Louise Hürlimann
    • 1
  • Lucia Arnold
    • 1
  • Nicolas U. Gerber
    • 1
  • Johannes Haybaeck
    • 4
  • Michel Mittelbronn
    • 5
  • Stefan Rutkowski
    • 2
  • Monika Hegi
    • 3
  • Michael A. Grotzer
    • 1
  1. 1.Neuro-Oncology ProgramUniversity Children’s HospitalZurichSwitzerland
  2. 2.Department of Pediatric Hematology and OncologyUniversity Medical Center Hamburg-EppendorfHamburgGermany
  3. 3.Laboratory of Brain Tumor Biology and Genetics, Department of Neurosurgery, BH-19-110Centre Hospitalier Universitaire Vaudois and University of LausanneLausanneSwitzerland
  4. 4.Institute of NeuropathologyUniversity Hospital of ZurichZurichSwitzerland
  5. 5.Institute of Neurology (Edinger Institute)Goethe-University FrankfurtFrankfurt/MainGermany

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