Mutation-Specific Antibodies for the Detection of EGFR Mutations in Non-Small-Cell Lung Cancer
J Yu, E Benedettini, DQ Li, T Gu, H Herebert, B Smith, R Polakiewicz, XM Zhou, M Loda, M Comb. Cell Signaling Technology, Inc, Danvers, MA; Dana-Farber Cancer Institute, Boston, MA; Second Xiangya Hosptial, Changsha, Hunan, China
Background: Activating mutations within the tyrosine kinase domain of the 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 the EGFR inhibitors Gefitinib and Erlotinib. The most common NSCLC-associated EGFR mutations are the 15 nucleotide in-frame deletion in exon 19 (E746-A750del) and the point mutation in exon 21 (L858R), together accounting for 90% of EGFR mutations. The ability to detect mutated gene products in cancer cells can identify patients most likely benefit from such therapies.
Design: We generated rabbit monoclonal antibodies (RmAb) against EGFR with E746-A750 deletions and L858R point mutation. We tested the antibodies by western blot, Immunofluorescence (IF) and immunohistochemistry (IHC) and used the antibodies staining 40 molecularly pre-typed tumors by IHC. Then, we used IHC by a panel of four antibodies (two EGFR mutation-specific antibodies, a control EGFR antibody and a pan-keratin antibody) to screen 340 cases of tumor samples with unknown genotype.
Results: The mutation-specific antibodies detect the corresponding mutant form of EGFR but not wild type EGFR by Western blotting, immunofluorescence (IF), and immunohistochemistry (IHC). IHC screening of a large panel of paraffin-embedded tumor samples of Non-Small-Cell Lung Cancer (NSCLC) patients shows that antibody reactivity is highly correlated with the presence of EGFR mutations.
DNA Sequencing and IHC Result of EGFR Mutation
|IHC||L858R (+)||dEGFR (+)||Wild Type||Failed|
Conclusions: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific and cost-effective methodology to identify lung cancer patients responsive to EGFR-based therapies.
Monday, March 9, 2009 8:45 AM
Platform Session: Section F, Monday Morning