Clinical Neuropathology practice news 1-2014: Pyrosequencing meets clinical and analytical performance criteria for routine testing of MGMT promoter methylation status in glioblastoma

Abstract

Testing of the MGMT promoter methylation status in glioblastoma is relevant for clinical decision making and research applications. Two recent and independent phase III therapy trials confirmed a prognostic and predictive value of the MGMT promoter methylation status in elderly glioblastoma patients. Several methods for MGMT promoter methylation testing have been proposed, but seem to be of limited test reliability. Therefore, and also due to feasibility reasons, translation of MGMT methylation testing into routine use has been protracted so far. Pyrosequencing after prior DNA bisulfite modification has emerged as a reliable, accurate, fast and easy-to-use method for MGMT promoter methylation testing in tumor tissues (including formalin-fixed and paraffin-embedded samples). We performed an intra- and inter-laboratory ring trial which demonstrates a high analytical performance of this technique. Thus, pyrosequencing-based assessment of MGMT promoter methylation status in glioblastoma meets the criteria of high analytical test performance and can be recommended for clinical application, provided that strict quality control is performed. Our article summarizes clinical indications, practical instructions and open issues for MGMT promoter methylation testing in glioblastoma using pyrosequencing.

Validation of ZAP-70 methylation and its relative significance in predicting outcome in chronic lymphocytic leukemia

“…20,21 For this validated assay in a third, clinical laboratory, the instrument used was a Pyromark ID (setting ID/MA0008) and 37 mL of PCR was sequenced. Also, nucleotides in the pyrosequencing run for the ID instrument were used undiluted, sepharose beads were increased from 2 mL to 3 mL, and sequencing primer was increased from 0.3 mM to 0.4 mM.…”

Radiomics signature: A potential biomarker for the prediction of MGMT promoter methylation in glioblastoma

“…Therefore, the detection of MGMT promoter methylation could help the selection of patients with GBM who may derive the most benefit from TMZ. The method in our study for detecting the MGMT promoter methylation status is pyrosequencing, which is one of the most accurate approaches . It is the gold standard for further building a correlation for learning the value of MRI.…”

“…The method in our study for detecting the MGMT promoter methylation status is pyrosequencing, which is one of the most accurate approaches. [23][24][25] It is the gold standard for further building a correlation for learning the value of MRI. Previous studies have indicated that the visual morphology of brain tumors such as mass effect, contrast enhancement, and edema is related to the genetic phenotype of brain tumors.…”

Gamma Knife Radiosurgery in Recurrent Glioblastoma

“…Cases with a mean methylation percentage of <8% were classified as MGMT promoter unmethylated and cases with a mean methylation percentage of ≥8% were classified as MGMT promoter methylated. [19,20]. Since pyrosequencing became available in our center in autumn 2013 and patients included in this study were all treated prior to 2013, MGMT promoter methylation results were not known at the time of treatment decision.…”

“…One 10-μm section of each specimen was used for MGMT promoter methylation analysis. MGMT pyrosequencing was performed as previously described by our group [19]: bisulfite modification was carried out using the Epi Tect Fast FFPE Bisulfite kit (Qiagen, Germany, article No. 59844).…”