title: Double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours.
abstract: BACKGROUND
  Our aim was to develop a new protocol for MGMT immunohistochemistry with good agreement between observers and good correlation with molecular genetic tests of tumour methylation. We examined 40 primary brain tumours (30 glioblastomas and 10 oligodendroglial tumours) with our new technique, namely double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumour antigens (CD34, CD45 and CD68). We compared the results with single-labelling immunohistochemistry for MGMT and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, a recognised molecular genetic technique which we applied as the gold-standard for the methylation status).
  
  
  RESULTS
  Double-labelling immunohistochemistry for MGMT produced a visual separation of tumourous and non-tumourous elements on the same histological slide, making it quick and easy to determine whether tumour cell nuclei were MGMT-positive or MGMT-negative (and thereby infer the methylation status of the tumour). We found good agreement between observers (kappa 0.76) and within observer (kappa 0.84). Furthermore, double-labelling showed good specificity (80%), sensitivity (73.33%), positive predictive value (PPV, 83.33%) and negative predictive value (NPV, 68.75%) compared to MS-MLPA. Double-labelling was quicker and easier to assess than single-labelling and it outperformed quantitative computerised image analysis of MGMT single-labelling in terms of sensitivity, specificity, PPV and NPV.
  
  
  CONCLUSIONS
  Double-labelling immunohistochemistry for MGMT and a cocktail of non-tumourous elements provides a "one look" method for determining whether tumour cell nuclei are MGMT-positive or MGMT-negative. This can be used to infer the methylation status of the tumour. There is good observer agreement and good specificity, sensitivity, PPV and NPV compared to a molecular gold-standard.
authors.affiliation: Cellular Pathology, St George's Hospital, Blackshaw Road, London SW17 0QT, UK. leslie.bridges@stgeorges.nhs.uk.
authors.forename: Elinor
authors.initials: E
authors.lastname: Burke
authors.forename: Mariana
authors.initials: M
authors.lastname: Grobler
authors.forename: Kay
authors.initials: K
authors.lastname: Elderfield
authors.forename: Frances
authors.initials: F
authors.lastname: Bond
authors.forename: Matthew
authors.initials: M
authors.lastname: Crocker
authors.forename: Rohan
authors.initials: R
authors.lastname: Taylor
authors.forename: Leslie R
authors.initials: LR
authors.lastname: Bridges
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D001932:Brain Neoplasms / Q000378:metabolism*; D015254:DNA Modification Methylases / Q000378:metabolism*; D045643:DNA Repair Enzymes / Q000378:metabolism*; D005188:False Negative Reactions; D005189:False Positive Reactions; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008745:Methylation; D009837:Oligodendroglioma / Q000378:metabolism*; D010784:Photomicrography; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D000097382:CD68 Molecule
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D015214:Antigens, Differentiation, Myelomonocytic; D015254:DNA Modification Methylases; D045643:DNA Repair Enzymes; D017493:Leukocyte Common Antigens; D025521:Tumor Suppressor Proteins; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C515491:MGMT protein, human; C516769:PTPRC protein, human