title: Comparative assessment of 5 methods (methylation-specific polymerase chain reaction, MethyLight, pyrosequencing, methylation-sensitive high-resolution melting, and immunohistochemistry) to analyze O6-methylguanine-DNA-methyltranferase in a series of 100 glioblastoma patients.
abstract: BACKGROUND
  There is a strong need to determine the best technique for O(6) -methylguanine-DNA-methyltranferase (MGMT) analysis, because MGMT status is currently used in clinical trials and occasionally in routine clinical practice for glioblastoma patients.
  
  
  METHODS
  The authors compared analytical performances and predictive values of 5 techniques in a series of 100 glioblastoma patients who received standard of care treatment (Stupp protocol).
  
  
  RESULTS
  MGMT promoter was considered methylated in 33%, 33%, 42%, and 60% of patients by methylation-sensitive high-resolution melting, MethyLight, pyrosequencing (with an optimal risk cutoff at 8% for the average percentage of the 5 CpGs tested), and methylation-specific polymerase chain reaction (MS-PCR), respectively. Fifty-nine percent of the samples had <23% (the optimal risk cutoff) of MGMT-positive tumor cells. The best predictive values for overall survival (OS), after adjustment for age and performance status, were obtained by pyrosequencing (hazard ratio [HR], 0.32; P < .0001), MS-PCR (HR, 0.37; P < .0001), and immunohistochemistry (HR, 0.43; P = .0005) as compared with methylation-sensitive high-resolution melting (HR, 0.52 P = .02) and MethyLight (HR, 0.6; P = .05). For progression-free survival (PFS), the best predictive values were obtained with pyrosequencing (HR, 0.35; P < .0001), methylation-sensitive high-resolution melting (HR, 0.46; P = .002), and MS-PCR (HR, 0.49; P = .002). Combining pyrosequencing and immunohistochemistry slightly improved predictive power for OS, but not for PFS. Poor reproducibility and interobserver variability were, however, observed for immunohistochemistry.
  
  
  CONCLUSIONS
  Good prediction of survival in addition to high reproducibility and sensitivity made pyrosequencing the best among the 5 techniques tested in this study.
authors.affiliation: Departement de Biologie, Centre Eugène Marquis, Rue de la Bataille Flandres Dunkerque, CS 44229, 35042 Rennes Cedex, France. v.quillien@rennes.unicancer.fr
authors.forename: Véronique
authors.initials: V
authors.lastname: Quillien
authors.forename: Audrey
authors.initials: A
authors.lastname: Lavenu
authors.forename: Lucie
authors.initials: L
authors.lastname: Karayan-Tapon
authors.forename: Catherine
authors.initials: C
authors.lastname: Carpentier
authors.forename: Marianne
authors.initials: M
authors.lastname: Labussière
authors.forename: Thierry
authors.initials: T
authors.lastname: Lesimple
authors.forename: Olivier
authors.initials: O
authors.lastname: Chinot
authors.forename: Michel
authors.initials: M
authors.lastname: Wager
authors.forename: Jérome
authors.initials: J
authors.lastname: Honnorat
authors.forename: Stephan
authors.initials: S
authors.lastname: Saikali
authors.forename: Frédéric
authors.initials: F
authors.lastname: Fina
authors.forename: Marc
authors.initials: M
authors.lastname: Sanson
authors.forename: Dominique
authors.initials: D
authors.lastname: Figarella-Branger
mesh_terms: D000328:Adult; D000368:Aged; D001932:Brain Neoplasms / Q000235:genetics; D019175:DNA Methylation; D005260:Female; D005909:Glioblastoma / Q000235:genetics* / Q000401:mortality; D006801:Humans; D008297:Male; D008875:Middle Aged; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000032:analysis / Q000235:genetics*; D015588:Observer Variation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011379:Prognosis; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016896:Treatment Outcome; D055815:Young Adult
chemical_list: D019853:O(6)-Methylguanine-DNA Methyltransferase