Molecular Diagnosis of Activating EGFR Mutations in Non–Small Cell Lung Cancer Using Mutation-Specific Antibodies for Immunohistochemical Analysis

Abstract

Purpose: Therapeutic responses of non-small cell lung carcinoma (NSCLC) to epidermal growth factor receptor (EGFR)-targeted drugs, such as gefitinib and erlotinib, are closely associated with activating EGFR mutations. The most common mutations are delE746-A750 in exon 19 and L858R in exon 21, accounting for ∼90% of all EGFR mutations. Recently, EGFR mutation-specific antibodies were developed and did well in immunohistochemical analysis, giving a sensitivity of ∼90%. We have investigated whether this method detects activating EGFR mutations with sensitivity comparable with direct DNA sequencing, which is used to detect these mutations in NSCLC.Experimental Design: We used antibodies specific for the E746-A750 deletion mutation in exon 19 and the L858R point mutation in exon 21 in Western blot analysis and immunohistochemistry to determine the presence of these mutations in NSCLC cell lines. We also examined these EGFR mutations in NSCLC tumor samples from 60 patients by immunohistochemically and direct DNA sequencing.Results: We were able to identify EGFR mutations in NSCLC tumor samples immunohistochemically with a sensitivity of 79% using the anti-delE746-A750 antibody and 83% using the anti-L858R antibody. Additional DNA sequencing markedly improved the sensitivity obtained by immunohistochemistry.Conclusions: This simple and rapid assay for detecting EGFR mutations, even in the small bronchial biopsies obtained in stage IV NSCLC patients, will be useful for diagnosing responsiveness to EGFRtargeted drugs in patients with NSCLC. Combining this with DNA sequencing is recommended for the development of improved personalized EGFR-targeted therapeutics.

High specificity but low sensitivity of mutation-specific antibodies against EGFR mutations in non-small-cell lung cancer

“…[14][15][16][17][18][19][20][21][22] All these studies used the same mutation-specific antibodies from Cell Signaling Technology and the methods were comparable. Simonetti et al found specificity of 100% for both antibodies in 78 unselected Caucasian patients.…”

“…21 Previous studies using the mutation-specific antibodies from Cell Signaling Technology have found sensitivity ranging from 23 to 80% for exon19 deletions and 36 to 100% for exon21 mutations. [14][15][16][17][18][19][20][21][22] These studies used different scores for intensity and cutoff point for positivity making comparisons between the individual studies difficult, and standardization is warranted. 23 No gold standard method is approved for detecting EGFR mutations in NSCLC.…”

“…Antibody dilutions tested were 1:50, 1:100, and 1:150 according to the literature. [14][15][16][17][18][19][20][21][22] The included sections were incubated with primary monoclonal rabbit antibodies against exon19 deletions (clone 6B6, Cell Signaling Technology, Boston, MA, USA) diluted to 1:150 (optimal dilution), against point mutation of exon21, L858R (clone 43B2, Cell Signaling Technology) diluted to 1:100 amplified by linker (optimal dilution), and with primary monoclonal mouse antibody against EGFR variantIII (clone 218C9, EGFR pharmDx kit, Dako, DK) in 20 min.…”

“…For these reasons, mutation-specific antibodies might be a relevant alternative for determining EGFR status in NSCLC. [14][15][16][17][18][19][20][21][22] So far, no standardized protocol exists for EGFR immunohistochemistry in these tumors. 9,23,24 The aim of this study was to determine sensitivity and specificity of mutation-specific antibodies in detection of EGFR mutations in an unselected cohort of Caucasian patients with NSCLC.…”

Standardization of Epidermal Growth Factor Receptor (EGFR) Measurement by Quantitative Immunofluorescence and Impact on Antibody-Based Mutation Detection in Non–Small Cell Lung Cancer

“…The sensitivity of the mutation-specific antibodies, although high, is not 100%, and after the initial reports, more recent studies have calculated the sensitivity to be as low as 80%. 37,38 In their cohort, Kitamura et al 39 were able to detect only 47% of mutations using the mutationspecific antibodies, probably because of the high frequency of nonclassic deletions in that particular cohort.…”