title: 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.
abstract: OBJECTIVE
  To analyze the reliability and reproducibility of PCR-based testing of O6-methylguanine-DNA methyltransferase gene (MGMT) promoter methylation status in formalin-fixed and paraffin-embedded (FFPE) neurosurgical biopsy specimens.
  
  
  METHODS
  We used 6 FFPE neurosurgical temporal lobe specimens of children and young adults with drug-resistant epilepsy. Histopathologically, all specimens showed CNS tissue with gliosis but no tumor tissue.
  
  
  METHODS
  For MGMT promoter methylation analysis, we used methylation specific PCR (MGMT MSP). In all 6 tissue specimens, 4 repetitive runs of MGMT MSP were performed.
  
  
  RESULTS
  We obtained conclusive results only in 13/24 (54.2%) MGMT MSP analyses. In 5/13 (38.5%) successful MSP runs, the results indicated presence of a methylated MGMT promoter. In only 1/6 specimens, MSP yielded consistent results in all 4 repetitive runs.
  
  
  CONCLUSIONS
  In our hands, MGMT MSP shows poor reliability and reproducibility of test results on FFPE neurosurgical tissue specimens. A more reliable method for diagnostic MGMT promoter methylation testing needs to be identified and validated by systematic testing of intra- and interlaboratory reliability and reproducibility. Alternatively, methods of tissue fixation that do not impair DNA quality but at the same time warrant high quality histopathology could facilitate molecular diagnostics.
authors.affiliation: Department of Internal Medicine I, Medical University, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Preusser
authors.forename: L
authors.initials: L
authors.lastname: Elezi
authors.forename: J A
authors.initials: JA
authors.lastname: Hainfellner
mesh_terms: D002648:Child; D019175:DNA Methylation / Q000502:physiology*; D015254:DNA Modification Methylases / Q000235:genetics / Q000378:metabolism*; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism*; D004827:Epilepsy / Q000378:metabolism / Q000473:pathology; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D013702:Temporal Lobe / Q000378:metabolism* / Q000473:pathology*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D055815:Young Adult
chemical_list: D005404:Fixatives; D025521:Tumor Suppressor Proteins; D005557:Formaldehyde; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes