TY - JOUR
T1 - A comparison of EGFR mutation testing methods in lung carcinoma
T2 - Direct sequencing, real-time PCR and immunohistochemistry
AU - Angulo, Bárbara
AU - Conde, Esther
AU - Suárez-Gauthier, Ana
AU - Plaza, Carlos
AU - Martínez, Rebeca
AU - Redondo, Pilar
AU - Izquierdo, Elisa
AU - Rubio-Viqueira, Belén
AU - Paz-Ares, Luis
AU - Hidalgo, Manuel
AU - López-Ríos, Fernando
N1 - Funding Information:
F. López-Ríos wishes to thank R. Franklin for her contribution to this work. Our thanks also go to the Tumour Bank at the ‘Laboratorio de Dianas Terapéuticas’, Hospital Universitario Madrid Sanchinarro, for handling the samples used in this study. We are most grateful to the Sequencing Service of the Genomic Unit at the Spanish National Cancer Centre (CNIO) for performing the direct sequencing assays. Some of the data in this manuscript were presented at the 13 World Conference of Lung Cancer, July 31-August 4, 2009, San Francisco, CA, USA. This work was partially funded by Fundación Mutua Madrileña and Fondo de Investigaciones Sanitarias (PIs: Esther Conde, Luis Paz-Ares and Fernando López-Ríos). DxS Limited provided some tests kits free of charge. Translated into English by Michelle Homden. th
Funding Information:
This work was partially funded by Fundación Mutua Madrileña and Fondo de Investigaciones Sanitarias (PIs: Esther Conde, Luis Paz-Ares and Fernando López-Ríos). DxS Limited provided some tests kits free of charge. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. Therascreen is a DxS Limited (now Qiagen) product. There are no other patents, marketed products, or products in development to declare.
PY - 2012/8/27
Y1 - 2012/8/27
N2 - 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.
AB - 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.
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U2 - 10.1371/journal.pone.0043842
DO - 10.1371/journal.pone.0043842
M3 - Article
C2 - 22952784
AN - SCOPUS:84865432696
SN - 1932-6203
VL - 7
JO - PloS one
JF - PloS one
IS - 8
M1 - e43842
ER -