The use of EGFR exon 19 and 21 unlabeled DNA probes to screen for activating mutations in non-small cell lung cancer.
Activating mutations in epidermal growth factor receptor-1 (EGFR) are found in 10-15% of Caucasian patients with non-small cell lung carcinoma (NSCLC). Approximately 90% of the mutations are deletions of several amino acids in exon 19 or point mutations in exon 21. Some studies suggest that these mutations identify patients that might benefit from targeted EGFR inhibitor therapy. DNA melting analysis of polymerase chain reaction products can screen for these mutations to identify this patient population. However, amplicon DNA melting analysis, although easily capable of detecting heterozygous mutations by heterodimer formation, becomes more difficult if mutations are homozygous or if the mutant allele is selectively amplified over wild type. Amplification of EGFR is common in NSCLC and this could compromise mutation detection by amplicon melting analysis. To overcome this potential limitation, we developed unlabeled, single-stranded DNA probes, complimentary to EGFR exon 19 and exon 21 where the common activating mutations occur. The unlabeled probes are incorporated into a standard polymerase chain reaction during the amplification of EGFR exons 19 and 21. The probe melting peak is easily distinguished from the amplicon melting peak, and probe melting is altered if mutations are present. This allows for easy identification of activating mutations even in homozygous or amplified states and is useful in the screening of NSCLC for the common EGFR activating mutations.
Willmore-Payne
Carlynn
C
Associated Regional and University Pathologists Institute for Clinical and Experimental Patholog y, Salt Lake City, UT 84108, USA. carlynn.willmore@aruplab.com
Holden
Joseph A
JA
Wittwer
Carl T
CT
Layfield
Lester J
LJ
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology; D001483:Base Sequence; D001709:Biotechnology; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods* / Q000706:statistics & numerical data; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics; D005091:Exons; D005260:Female; D018773:Genes, erbB-1; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008875:Middle Aged; D015336:Molecular Probe Techniques / Q000706:statistics & numerical data; D009154:Mutation; D012680:Sensitivity and Specificity; D017384:Sequence Deletion; D015533:Transcriptional Activation
D017931:DNA Primers; D015342:DNA Probes; D004273:DNA, Neoplasm