title: A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry.
abstract: The objective of this study is to compare two EGFR testing methodologies (a commercial real-time PCR kit and a specific EGFR mutant immunohistochemistry), with direct sequencing and to investigate the limit of detection (LOD) of both PCR-based methods. We identified EGFR mutations in 21 (16%) of the 136 tumours analyzed by direct sequencing. Interestingly, the Therascreen EGFR Mutation Test kit was able to characterize as wild-type one tumour that could not be analyzed by direct sequencing of the PCR product. We then compared the LOD of the kit and that of direct sequencing using the available mutant tumours. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in nine of the 18 tumours (50%), which tested positive with the real-time quantitative PCR method. In all cases, EGFR mutation was identified at a dilution of 5%. Where the mutant DNA represented 30% of the total DNA, sequencing was able to detect mutations in 12 out of 19 cases (63%). Additional experiments with genetically defined standards (EGFR ΔE746-A750/+ and EGFR L858R/+) yielded similar results. Immunohistochemistry (IHC) staining with exon 19-specific antibody was seen in eight out of nine cases with E746-A750del detected by direct sequencing. Neither of the two tumours with complex deletions were positive. Of the five L858R-mutated tumours detected by the PCR methods, only two were positive for the exon 21-specific antibody. The specificity was 100% for both antibodies. The LOD of the real-time PCR method was lower than that of direct sequencing. The mutation specific IHC produced excellent specificity.
authors.affiliation: Laboratorio de Dianas Terapéuticas, Faculty of Medicine, Centro Integral Oncológico Clara Campal, Hospital HM Universitario Sanchinarro, Universidad San Pablo-CEU, Madrid, Spain.
authors.forename: Bárbara
authors.initials: B
authors.lastname: Angulo
authors.forename: Esther
authors.initials: E
authors.lastname: Conde
authors.forename: Ana
authors.initials: A
authors.lastname: Suárez-Gauthier
authors.forename: Carlos
authors.initials: C
authors.lastname: Plaza
authors.forename: Rebeca
authors.initials: R
authors.lastname: Martínez
authors.forename: Pilar
authors.initials: P
authors.lastname: Redondo
authors.forename: Elisa
authors.initials: E
authors.lastname: Izquierdo
authors.forename: Belén
authors.initials: B
authors.lastname: Rubio-Viqueira
authors.forename: Luis
authors.initials: L
authors.lastname: Paz-Ares
authors.forename: Manuel
authors.initials: M
authors.lastname: Hidalgo
authors.forename: Fernando
authors.initials: F
authors.lastname: López-Ríos
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D057230:Limit of Detection; D008175:Lung Neoplasms / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: C512478:EGFR protein, human; D066246:ErbB Receptors