Detection of EGFR Mutations and Exon 19 Deletions Using Primer Extension and Fragment Analysis in a Single Reaction.
Eric Olsen, Javier Mestas, Wayne Grody, Josh Deignan. University of California, Los Angeles
Background: The epidermal growth factor receptor (EGFR) is a cellular transmembrane receptor with tyrosine kinase activity that takes part in human cancer. EGFR -dependent signaling is involved in cancer cell proliferation, invasion and metastasis. Targeting EGFR is a directed molecular approach in cancer therapy. EGFR inhibitors are being utilized in the chemotherapy regimens of patients with advanced non-small cell lung cancers. Here, we have developed an assay in which we can determine the mutational status of EGFR in a single multiplex reaction using single-base extension and fragment analysis.
Design: Genomic DNA samples were prepared from formalin-fixed, paraffin-embedded lung tumor biopsies from the Department of Pathology at UCLA Medical Center. Analysis of the most common mutations located within exons 18, 20 and 21 of the EGFR gene, as well as the deletions within exon 19, was performed in a single tube by amplifying small regions flanking the mutation sites in a multiplex polymerase chain reaction (PCR). In order to determine the identity of the nucleotides at the SNP site, allele-specific fluorescently-labeled oligonucleotides were generated in a single-base extension reaction using the ABI PRISM® SNaPshot™ Multiplex Kit, and detected by capillary electrophoresis. Fragment analysis of the exon 19 deletion mutation in the same reaction was made possible by using fluorescently-labeled amplification primers for exon 19 during the multiplex PCR.
Results: The EGFR mutation detection assay was capable of detecting the G719S mutation in exon 18, the deletions in exon 19, the T790M mutation in exon 20, and the L858R and L861Q mutations in exon 21. The accuracy of this assay was determined by method comparison with Sanger sequencing and was found to be 100% for this limited sample. The analytical sensitivity of the EGFR mutation detection assay was approximately 1 ng of genomic DNA and the inter-assay and intra-assay precisions were both 100%. DNA template amounts up to 1 µg per reaction were shown not to interfere with the assay and the analytical sensitivity of the assay was determined to be ∼10% mutant DNA in a wildtype background.
Conclusions: EGFR testing can be completed efficiently using a combination of primer extension and fragment analysis reactions which are relatively inexpensive to perform. This assay is capable of identifying EGFR mutations associated with a sensitivity or resistance to tyrosine kinase inhibitors. Such molecular information can help direct management of care in patients with advanced lung cancer.
Wednesday, March 2, 2011 9:30 AM
Poster Session V # 246, Wednesday Morning