MGMT Pyrosequencing Methylation Assay for Glioblastoma
Polymerase Chain Reaction (PCR)
Formalin-fixed, paraffin-embedded block
Formalin-fixed, paraffin-embedded block
Positive for MGMT methylation: ≥ of 10% of any CpG island or average of all 5 CpG islands.
Negative for MGMT methylation: >10% of methylation or average of all CpG islands analyzed.
Glioblastoma is the most common and most aggressive malignant primary brain tumor. While occurring in only two to three cases per 100,000 people in North America, glioblastoma represents 52% of all functional tissue brain tumor cases and 20% of all intracranial tumors. Prognosis for those diagnosed with glioblastoma is poor, with a median survival time of about 14 months.1
Patients with glioblastoma can be treated with alkylating agents, such as Temador® (temozolomide). Epigenetic silencing of the MGMT (O6-methylguanine-DNA methyltransferase) DNA-repair gene by promoter methylation compromises DNA repair and has been associated with longer survival in patients with glioblastoma who receive temozolomide.2,3
Temozolomide kills tumor cells by producing cross-links between DNA strands and inhibiting DNA replication. The most common alkylation site is the O6 position of guanine. O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that reverses such DNA alkylation and confers chemoresistance by repairing DNA damage. Temozolomide seems to work by sensitizing the tumor cells to radiation.4
Recent clinical studies confirm that the presence of MGMT promoter methylation in tumor samples corresponds to an increased likelihood that tumor cells would be responsive to temozolomide.3,4,5,6 If the promotor was methylated, temozolomide was more effective. It is estimated that approximately 40 to 50% of glioblastoma tumors exhibit MGMT gene methylation, which correlates significantly with reduced DNA damage repair induced by alkylating agents and significantly enhanced chemosensitivity.4
According to recent clinical trials, glioblastoma patients with MGMT methylation respond to temozolomide two to three times better than those lacking of MGMT methylation. Prolonged overall and progression-free survival at 24 months was 80% for those with MGMT methylation vs. 20% for those lacking MGMT methylation.
Diagnostic MGMT testing requires sufficient and optimally preserved tumor tissue. Cleveland Clinic’s MGMT methylation assay is a new, quantitative MSP test to detect the methylation status of brain tissue that has undergone thorough clinical evaluation. Our protocol calls for a minimal tissue sample size of ½-centimeter, which is much smaller than other laboratories that typically require at least a minimum 1-centimeter sample size. The best results are obtained with cryopreserved tumor specimens.
For patients diagnosed with glioblastoma to determine if a methylated MGMT promoter is present, which is a favorable prognostic indicator for temozolomide treatment. Individuals without a methylated MGMT promoter do not have such a benefit.
MGMT “silence” is the most significant guide for the treatment of glioblastoma. This assay is to validate the methylation status of the MGMT gene.
Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. Real-time sequence information is highly suitable for quantification of CpG methylation. We have validated pyrosequencing-based assay in detection of MGMT methylation in paraffin-embedded biopsy tissue specimens. With 10% average methylation as a cutoff, MGMT promoter methylation was detected in glioblastoma, but not detected in non-neoplastic brain tissue. The analytical sensitivity of the assay is 5% of target cells harboring MGMT methylation.
Diagnostic MGMT testing requires sufficient and optimally preserved tumor tissue. The biopsy should be at least 0.5 cm in size and necrosis should be less than 15%. Both frozen and paraffin-embedded tissue are suitable for the pyrosequencing-based MGMT methylation assay.
Positive for MGMT methylation:
Equal or greater than 10% of methylation in any CpG island or in average of all CpG islands analyzed.
Negative for MGMT methylation:
Less than 10% of any CpG island or in average of all CpG islands analyzed.
1. Van Meir EG, et al. “Exciting New Advances in NeuroOncology: The Avenue to a Cure for Malignant Glioma”. CA: A Cancer Journal for Clinicians. 60 (3):166–93. doi:10.3322/caac.20069. PMC 2888474. PMID 20445000. 2010.
2. Stupp R, et al. Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma. N Engl J Med. 352:987-996.
3. Hegi M, et al. MGMT Gene Silencing and Benefit from Temozolomide in Glioblastoma. N Engl J Med. 2005;352;10:997-1003.
4. Chamberlain Marc C, et al. “Early necrosis following concurrent Temodar and radiotherapy in patients with glioblastoma”. Journal of Neuro-Oncology. 82(1):81-3. doi:10.1007/s11060-006-9241-y. PMID 16944309.
5. Vlassenbroeck I, et al. Validation of Real-Time MethylationSpecific PCR to Determine O(6)-Methylguanine-DNA Methyl-transferase Gene Promoter Methylation in Glioma. J. Molecular Diagnostics. 2008;10;4:332-337.
6. Brandes A, et al. MGMT Promoter Methylation Status Can Predict the Incidence and Outcome of Pseudoprogression After Concomitant Radiochemotheraphy in Newly Diagnosed Glioblastoma Patients. J. Clinical Oncology. 2008; 26;13:2192-2197.