title: A simple quantitative diagnostic alternative for MGMT DNA-methylation testing on RCL2 fixed paraffin embedded tumors using restriction coupled qPCR.
abstract: BACKGROUND
  MGMT promoter methylation is associated with favorable prognosis and chemosensitivity in glioblastoma multiforme (GBM), especially in elderly patients. We aimed to develop a simple methylation-sensitive restriction enzyme (MSRE)-based quantitative PCR (qPCR) assay, allowing the quantification of MGMT promoter methylation.
  
  
  METHODS
  DNA was extracted from non-neoplastic brain (n = 24) and GBM samples (n = 20) upon 3 different sample conservation conditions (-80 °C, formalin-fixed and paraffin-embedded (FFPE); RCL2-fixed). We evaluated the suitability of each fixation method with respect to the MSRE-coupled qPCR methylation analyses. Methylation data were validated by MALDITOF.
  
  
  RESULTS
  qPCR was used for evaluation of alternative tissue conservation procedures. DNA from FFPE tissue failed reliable testing; DNA from both RCL2-fixed and fresh frozen tissues performed equally well and was further used for validation of the quantitative MGMT methylation assay (limit of detection (LOD): 19.58 pg), using individual's undigested sample DNA for calibration. MGMT methylation analysis in non-neoplastic brain identified a background methylation of 0.10 ± 11% which we used for defining a cut-off of 0.32% for patient stratification. Of GBM patients 9 were MGMT methylationpositive (range: 0.56 - 91.95%), and 11 tested negative. MALDI-TOF measurements resulted in a concordant classification of 94% of GBM samples in comparison to qPCR.
  
  
  CONCLUSIONS
  The presented methodology allows quantitative MGMT promoter methylation analyses. An amount of 200 ng DNA is sufficient for triplicate analyses including control reactions and individual calibration curves, thus excluding any DNA qualityderived bias. The combination of RCL2-fixation and quantitative methylation analyses improves pathological routine examination when histological and molecular analyses on limited amounts of tumor samples are necessary for patient stratification.
authors.forename: Walter
authors.initials: W
authors.lastname: Pulverer
authors.forename: Manuela
authors.initials: M
authors.lastname: Hofner
authors.forename: Matthias
authors.initials: M
authors.lastname: Preusser
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Dirnberger
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Hainfellner
authors.forename: Andreas
authors.initials: A
authors.lastname: Weinhaeusel
mesh_terms: D000328:Adult; D000368:Aged; D001932:Brain Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D016022:Case-Control Studies; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000378:metabolism*; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D005404:Fixatives; D005557:Formaldehyde; D005909:Glioblastoma / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008875:Middle Aged; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D011401:Promoter Regions, Genetic; D016707:Tissue Fixation / Q000379:methods*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D005404:Fixatives; D025521:Tumor Suppressor Proteins; D005557:Formaldehyde; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D004262:DNA Restriction Enzymes; D045643:DNA Repair Enzymes