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MGMT Testing (Glioblastoma)

The copy number of methylated O6-Methylguanine-DNA Methyltransferase (MGMT) is determined relative to the copy number of beta-actin (ACTB) by real-time methylation specific polymerase chain reaction (MSP). In order to do so, tumor tissue content is enriched by manual microdissection followed by DNA extraction. DNA is quantified using Picogreen and modified by bisulfite treatment which causes unmethylated cytosines to be converted to uracyl while methylated cytosines remain unchanged. Subsequently, a real-time Methylation Specific PCR (MSP) is performed on the Quantstudio 5 instrument to quantify the copy number of MGMT and ACTB (1). This technique consists of performing a PCR with real-time detection using the amplifluor detection system. Samples with an unknown copy number can be quantified by comparison of their Ct values with those of a plasmid standard curve.

Clinical Implication

MGMT is a DNA repair enzyme that is frequently deactivated by hypermethylation in human cancer. When cells lack MGMT activity, DNA damage is not repaired properly making an individual more prone to the development of cancer. Of interest, this lack of MGMT activity also makes a tumor cell more sensitive to radiation therapy and certain alkylating drugs such as temozolomide (2). In the international prognostic Phase III (RTOG 0525) validation study, the MGMT assay successfully identified newly diagnosed glioblastoma patients who are more likely to live longer and have a longer progression free time period following treatment with temozolomide (3-5). Recently, a new publication was released in which both methylated and “grey zone” patients performed significantly better for overall survival than truly unmethylated patients (6).

Specimen Requirements

Acceptable specimens for the assay are formalin-fixed, paraffin-embedded glioma tissue specimens with a fixation time of 6-48 hours.


One representative paraffin block is preferred. Alternatively, 5 unstained tissue sections are accepted (1 slide of 5 µm thickness for H&E staining and a minimum of 4 slides of 5-10 µm thickness for MGMT testing).

Storage and Shipment Instructions

Maintain and ship specimens at ambient temperature.


For some samples, an invalid result will be obtained due to an insufficient number of ACTB copies in combination with a number of MGMT copies below the limit of detection. Mostly, this is explained by insufficient DNA quantity and/or quality. For invalid samples with a copy number for MGMT and/or ACTB around the LOD/LOQ and cut-off, it is recommended to perform a retest starting from the bisulfite treated DNA. Fixatives other than formalin or prolonged fixation time may give rise to inadequate results.

Special Requirements


Turn-Around Time

Five to 7 business days for slides and paraffin blocks respectively.


  1. Vlassenbroeck I et al. Validation of Real-Time Methylation-Specific PCR to determine O6-Methylguanine-DNA Methyltransferase Gene Promotor Methylation in Glioma. J Mol Diagn. 2008 Jul;10(4):332-7
  2. Hegi ME et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 2005 Mar 10;352(10):997-1003.
  3. Gilbert MR et al. Dose-dense temozolomide for newly diagnosed glioblastoma: a randomized phase III clinical trial.(7). J Clin Oncol. 2013 Nov 10;31(32):4085-91.
  4. Malmström A et al. Temozolomide versus standard 6-week radiotherapy versus hypofractionated radiotherapy in patients older than 60 years with glioblastoma: the Nordic randomised, phase 3 trial. Lancet Oncol 2012 Sep; 13(9): 916-926.
  5. Wick W et al. Temozolomide chemotherapy alone versus radiotherapy alone for malignant astrocytoma in the elderly: the NOA-08 randomised, phase 3 trial. Lancet Oncol 2012 Jul; 13(7): 707-715.
  6. Hegi ME et al. MGMT Promoter Methylation Cutoff with Safety Margin for Selecting Glioblastoma Patients into Trials Omitting Temozolomide. A Pooled Analysis of Four Clinical Trials. Clin Cancer Res 2019 Mar 15; 25(6): 1809-1816.

Updated on 23 May 2019