Abstract
Epigenetic silencing of the MGMT gene through promoter methylation correlates with improved survival in Glioblastoma Multiforme (GBM) patients receiving concurrent chemoradiotherapy. Although the clinical benefit is primarily seen in patients with methylated MGMT promoter, some unmethylated patients also respond to Temozolomide. One possible explanation may be intratumoral heterogeneity. This study was designed to assess the methylation status of the MGMT promoter in different areas of GBM and determine if methylation status varied depending on the fixation technique (paraffin-embedding versus fresh frozen) used to store tissue. Using intraoperative navigation, biopsies were obtained from three distinct regions: the enhancing outer area, the non-enhancing inner core, and an area immediately outside the enhancing region. Only patients with GBM were included for evaluation and analysis. Samples taken from each area were divided with half stored by flash freezing and the other half stored using paraffin fixation. Methylation Specific-PCR (MS-PCR) was used for analysis of MGMT promoter methylation. Thirteen patients were included. Ten were male with a median age of 62 years. In each patient, samples were taken from the enhancing rim and the necrotic centre. However, it was not considered safe or feasible to obtain samples from the area immediately adjacent to the enhancing tumor rim in one case. All patients were homogeneous for methylation status throughout their tumor and tissue taken adjacent to it when frozen tissue was used. However, four patients had discrepancies in the MGMT promoter status between the frozen and paraffin-embedded blocks and one patient was not homogeneous within the tumor when paraffin-embedded tissue was used. MGMT promoter methylation status was homogeneous in all GBM tumors. Our observation that methylation status varied depending if the DNA was extracted from paraffin-embedded versus frozen tissue is concerning. Although the reason for this is unclear, we postulate that the timing from resection to fixation or the process of fixation itself may potentially alter methylation status in paraffin-embedded tumors.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Stupp R, Mason WP, van den Bent MJ et al (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996
van Rijn J, Heimans JJ, van den Berg J, van der Valk P, Slotman BJ (2000) Survival of human glioma cells treated with various combination of temozolomide and X-rays. Int J Radiat Oncol Biol Phys 47:779–784
Wedge SR, Porteous JK, Glaser MG, Marcus K, Newlands ES (1997) In vitro evaluation of temozolomide combined with X-irradiation. Anticancer Drugs 8:92–97
Wick W, Wick A, Schulz JB, Dichgans J, Rodemann HP, Weller M (2002) Prevention of irradiation-induced glioma cell invasion by temozolomide involves caspase 3 activity and cleavage of focal adhesion kinase. Cancer Res 62:1915–1919
Hegi ME, Diserens AC, Gorlia T et al (2005) MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352:997–1003
Liu L, Markowitz S, Gerson SL (1996) Mismatch repair mutations override alkyltransferase in conferring resistance to temozolomide but not to 1,3-bis(2-chloroethyl)nitrosourea. Cancer Res 56:5375–5379
Ochs K, Kaina B (2000) Apoptosis induced by DNA damage O6-methylguanine is bcl-2 and caspase-9/3 regulated and Fas/caspase-8 independent. Cancer Res 60:5815–5824
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 343:1350–1354
Hegi ME, Diserens AC, Godard S et al (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
Juillerat-Jeanneret L, Bernasconi CC, Bricod C et al (2008) Heterogeneity of human glioblastoma: Glutathione-S-transferase and methylguanine-methyltransferase. Cancer Invest 26:597–609
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 87:71–78
Srinivasan M, Sedmak D, Jewell S (2002) Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol 161:1961–1971
Talaulikar D, Gray JX, Shadbolt B, McNiven M, Dahlstrom JE (2008) A comparative study of the quality of DNA obtained from fresh frozen and formalin-fixed decalcified paraffin-embedded bone marrow trephine biopsy specimens using two different methods. J Clin Pathol 61:119–123
Cao VT, Jung TY, Jung S et al (2009) The correlation and prognostic significance of MGMT promoter methylation and MGMT protein in glioblastomas. Neurosurgery 65:866–875
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 121:2458–2464
Nakagawa T, Ido K, Sakuma T, Takeuchi H, Sato K, Kubota T (2009) Prognostic significance of the immunohistochemical expression of O(6)-methylguanine-DNA methyltransferase, P-glycoprotein, and multidrug resistance protein-1 in glioblastomas. Neuropathology 29:379–388
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 33:469–475
Preusser M, Elezi L, Hainfellner JA (2008) Reliability and reproducibility of PCR-based testing of O6-methylguanine-DNA methyltransferase gene (MGMT) promoter methylation status in formalin-fixed and paraffin-embedded neurosurgical biopsy specimens. Clin Neuropathol 27:388–390
Wller M, Stupp R, Reifenberger G et al (2010) MGMT promoter methylation in malignant gliomas: ready for personalized medicine? Nat Rev Neurol 6:39–51
Cilengitide for subjects with newly diagnosed glioblastoma multiforme and methylated mgmt gene promoter—a multicenter, open-label, controlled phase III study, testing cilengitide in combination with standard treatment (temozolomide with concomitant radiation therapy, followed by temozolomide maintenance therapy) versus standard treatment alone. http://www.clinicaltrials.gov/ct2/show/NCT00689221. Accessed 16 Feb 2010
Cilengitide in subjects with newly diagnosed glioblastoma multiforme and unmethylated mgmt gene promoter—a multicenter, open-label phase ii study, investigating two cilengitide regimens in combination with standard treatment (temozolomide with concomitant radiation therapy, followed by temozolomide maintenance therapy). http://www.clinicaltrials.gov/ct2/show/NCT00813943. Accessed 16 Feb 2010
Acknowledgments
We would like to acknowledge Elizabeth Kornaga, Angela Chan, Jaclyn Vande Graaf and Annabelle Mastalic for their collaboration in this study. This study was supported in part by an Alberta Cancer Board Research Legacy Grant and the Brain Tumor Foundation of Canada.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
11060_2010_307_MOESM1_ESM.tif
Verification of patient 8 FFPE MGMT-MSP result: Figure 1a shows the original result for patient 8 FFPE tissues. Figure 1b is the result from an independent experiment using DNA re-extracted from FFPE tissues B, C and D in order to rule out possible PCR contamination. NTC = no template control, U87MG = methylation positive control, HTB30 = methylation negative control 1 (TIFF 907 kb)
Rights and permissions
About this article
Cite this article
Hamilton, M.G., Roldán, G., Magliocco, A. et al. Determination of the methylation status of MGMT in different regions within glioblastoma multiforme. J Neurooncol 102, 255–260 (2011). https://doi.org/10.1007/s11060-010-0307-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11060-010-0307-5