Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas - PubMed (original) (raw)

Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas

Qiulu Pan et al. J Mol Diagn. 2005 Aug.

Abstract

Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene are present in lung adenocarcinomas that respond to the EGFR inhibitors gefitinib and erlotinib. Two types of mutations account for approximately 90% of mutated cases: short in-frame deletions in exon 19 and a specific point mutation in exon 21 at codon 858 (L858R). Screening for these mutations has been based mainly on direct sequencing. We report here the development and validation of polymerase chain reaction-based assays for these two predominant types of EGFR mutations. The assay for exon 19 mutations is based on length analysis of fluorescently labeled polymerase chain reaction products, and the assay for the exon 21 L858R mutation is based on a new Sau96I restriction site created by this mutation. Using serial dilutions of DNAs from lung cancer cell lines harboring either exon 19 or 21 mutations, we detected these mutations in the presence of up to approximately 90% normal DNA. In a test set of 39 lung cancer samples, direct sequencing detected mutations in 25 cases whereas our assays were positive in 29 cases, including 4 cases in which mutations were not apparent by sequencing. These assays offer higher sensitivity and ease of scoring and eliminate the need for sequencing, providing a robust and accessible approach to the rapid identification of most lung cancer patients likely to respond to EGFR inhibitors.

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Figures

Figure 1

Figure 1

EGFR exon 21 L858R mutation assay design showing primer locations and native and mutant Sau96I restriction enzyme sites.

Figure 2

Figure 2

EGFR exon 19 deletion assay: examples of detection of 9-, 12-, 15-, and 18-bp deletions in EGFR exon 19. The variable relative heights of the deleted and germline peaks may reflect different proportions of tumor cells in these clinical tumor DNAs, or different levels of copy number alterations in the mutated EGFR allele.

Figure 3

Figure 3

Sensitivity analysis of EGFR exon 19 deletion assay using serial dilutions of H1650 cell line DNA into normal DNA. Note that for dilutions of 25% and less the scale has been adjusted to highlight the mutant peak.

Figure 4

Figure 4

EGFR exon 21 L858R mutation assay: examples of undigested, digested germline, and digested mutant PCR products.

Figure 5

Figure 5

Sensitivity analysis of EGFR exon 21 L858R mutation assay using serial dilutions of H1975 cell line DNA into normal DNA. Note that for dilutions of 12.5% and less the scale has been adjusted to highlight the mutant peak.

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