Purpose: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10-20% 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 (RmAb) against each of these two most common EGFR mutations, and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry (IHC). Experimental Design: We tested mutation-specific antibodies by Western blot, Immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by IHC 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 IHC 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 IHC assay is 92%, with a specificity of 99% as compared to direct and MS-based DNA sequencing. Conclusions: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific and costeffective methodology to identify lung cancer patients responsive to EGFR-based therapies.

Mutation-Specific Antibodies for the Detection of EGFR Mutation in Non-Small-Cell Lung Cancer / J. Yu, S. Kane, J. Wu, E. Benedettini, D. Li, C. Reeves, G. Innocenti, R. Wetzel, K. Crosby, A. Becker, M. Ferrante, W.G. Cheung, X. Hong, L.R. Chirieac, L.M. Sholl, H. Haack , B. Smith, R. Polakiewicz, Y. Tan, T. Gu, M. Loda, X. Zhou, M. Comb. - In: CLINICAL CANCER RESEARCH. - ISSN 1078-0432. - 15:9(2009 Apr), pp. 3023-3028. [10.1158/1078-0432.CCR-08-2739]

Mutation-Specific Antibodies for the Detection of EGFR Mutation in Non-Small-Cell Lung Cancer

E. Benedettini;
2009-04

Abstract

Purpose: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10-20% 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 (RmAb) against each of these two most common EGFR mutations, and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry (IHC). Experimental Design: We tested mutation-specific antibodies by Western blot, Immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by IHC 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 IHC 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 IHC assay is 92%, with a specificity of 99% as compared to direct and MS-based DNA sequencing. Conclusions: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific and costeffective methodology to identify lung cancer patients responsive to EGFR-based therapies.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/64035
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