A comparison of EGFR mutation testing methods in lung carcinoma: Direct sequencing, real-time PCR and immunohistochemistry

Bárbara Angulo, Esther Conde, Ana Suárez-Gauthier, Carlos Plaza, Rebeca Martínez, Pilar Redondo, Elisa Izquierdo, Belén Rubio-Viqueira, Luis Paz-Ares, Manuel Hidalgo, Fernando López-Ríos

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article numbere43842
JournalPLoS One
Volume7
Issue number8
DOIs
StatePublished - Aug 27 2012
Externally publishedYes

Fingerprint

carcinoma
immunohistochemistry
Tumors
Real-Time Polymerase Chain Reaction
quantitative polymerase chain reaction
Immunohistochemistry
lungs
Carcinoma
mutation
Lung
Mutation
neoplasms
Testing
Limit of Detection
Neoplasms
detection limit
testing
Polymerase Chain Reaction
mutants
Dilution

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Angulo, B., Conde, E., Suárez-Gauthier, A., Plaza, C., Martínez, R., Redondo, P., ... López-Ríos, F. (2012). A comparison of EGFR mutation testing methods in lung carcinoma: Direct sequencing, real-time PCR and immunohistochemistry. PLoS One, 7(8), [e43842]. https://doi.org/10.1371/journal.pone.0043842

A comparison of EGFR mutation testing methods in lung carcinoma : Direct sequencing, real-time PCR and immunohistochemistry. / Angulo, Bárbara; Conde, Esther; Suárez-Gauthier, Ana; Plaza, Carlos; Martínez, Rebeca; Redondo, Pilar; Izquierdo, Elisa; Rubio-Viqueira, Belén; Paz-Ares, Luis; Hidalgo, Manuel; López-Ríos, Fernando.

In: PLoS One, Vol. 7, No. 8, e43842, 27.08.2012.

Research output: Contribution to journalArticle

Angulo, B, Conde, E, Suárez-Gauthier, A, Plaza, C, Martínez, R, Redondo, P, Izquierdo, E, Rubio-Viqueira, B, Paz-Ares, L, Hidalgo, M & López-Ríos, F 2012, 'A comparison of EGFR mutation testing methods in lung carcinoma: Direct sequencing, real-time PCR and immunohistochemistry', PLoS One, vol. 7, no. 8, e43842. https://doi.org/10.1371/journal.pone.0043842
Angulo, Bárbara ; Conde, Esther ; Suárez-Gauthier, Ana ; Plaza, Carlos ; Martínez, Rebeca ; Redondo, Pilar ; Izquierdo, Elisa ; Rubio-Viqueira, Belén ; Paz-Ares, Luis ; Hidalgo, Manuel ; López-Ríos, Fernando. / A comparison of EGFR mutation testing methods in lung carcinoma : Direct sequencing, real-time PCR and immunohistochemistry. In: PLoS One. 2012 ; Vol. 7, No. 8.
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