Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer.
OBJECTIVE
Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non-small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLC-associated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry.


METHODS
We tested mutation-specific antibodies by Western blot, immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by immunohistochemistry with these antibodies. Finally, with a panel of four antibodies, we screened a large set of NSCLC patient samples with unknown genotype and confirmed the immunohistochemistry results by DNA sequencing.


RESULTS
These two antibodies specifically detect the corresponding mutant form of EGFR by Western blotting, immunofluorescence, and immunohistochemistry. Screening a panel of 340 paraffin-embedded NSCLC tumor samples with these antibodies showed that the sensitivity of the immunohistochemistry assay is 92%, with a specificity of 99% as compared with direct and mass spectrometry-based DNA sequencing.


CONCLUSIONS
This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific, and cost-effective method to identify lung cancer patients responsive to EGFR-based therapies.
Yu
Jian
J
Cell Signaling Technology, Inc., Danvers, Massachusetts, USA.
Kane
Susan
S
Wu
Jiong
J
Benedettini
Elisa
E
Li
Daiqiang
D
Reeves
Cynthia
C
Innocenti
Gregory
G
Wetzel
Randy
R
Crosby
Katherine
K
Becker
Alison
A
Ferrante
Michelle
M
Cheung
Wan Cheung
WC
Hong
Xiqiang
X
Chirieac
Lucian R
LR
Sholl
Lynette M
LM
Haack
Herbert
H
Smith
Bradley L
BL
Polakiewicz
Roberto D
RD
Tan
Yi
Y
Gu
Ting-Lei
TL
Loda
Massimo
M
Zhou
Xinmin
X
Comb
Michael J
MJ
D000818:Animals; D000911:Antibodies, Monoclonal; D001681:Biological Assay; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000556:secondary; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics*; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000276:immunology; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D009154:Mutation / Q000276:immunology*; D011817:Rabbits; D012680:Sensitivity and Specificity; D017384:Sequence Deletion; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D007074:Immunoglobulin G; C512478:EGFR protein, human; D066246:ErbB Receptors