Abstract
BackgroundTreatment decisions in advanced non-small cell lung cancer rely on accurate analysis of the EGFR mutation status in small tissue samples. Sanger sequencing of PCR products is unbiased and cheap, but its detection threshold requiring 20 % infiltration by malignant cells is not optimal. Commercial kits, based on quantitative real-time PCR have better detection limits and can detect a wide spectrum of mutations but are considerably more expensive.MethodsWe developed a wild-type blocking PCR for EGFR G719A/S/C (exon 18), exon 19 deletions, and exon 20 insertions using locked nucleic acid (LNA) probes. The amplification products of positive reactions were analyzed by Sanger sequencing. We retrospectively validated this assay by comparison of the EGFR mutation status as obtained with Fragment Length Analysis and the Therascreen EGFR RGQ PCR kit.ResultsThe EGFR mutation status for exon 18 and 19 as obtained with the LNA-PCR/sequencing assay correlated adequately with the results obtained by the other independent methods. Due to the lack of structural consistency among the insertions in exon 20, the latter are less amenable for a LNA-PCR design.ConclusionsThe LNA-PCR/sequencing assay presented here is specific, sensitive, and has a low detection threshold. In combination with allele-specific PCR reactions for T790M (exon 20) and L858R (exon 21), a wider scope of EGFR mutations can be assessed at a lower cost.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1272520418142748
Highlights
Treatment decisions in advanced non-small cell lung cancer rely on accurate analysis of the Epidermal Growth Factor Receptor (EGFR) mutation status in small tissue samples
We developed an assay for EGFR mutations G719A/S/C in exon and deletions in exon based on a locked nucleic acid (LNA) wild-type blocking PCR followed by Sanger sequencing of the amplicon
The most common EGFR mutations are small in-frame deletions in exon 19 and the L858R point mutation in exon 21 [4, 5]. They account for about 90 % of all EGFR mutations reported in lung adenocarcinoma and are known to be sensitive to the EGFR-tyrosine kinase inhibitor (TKI) gefitinib, erlotinib, or afatinib [7,8,9,10]
Summary
Treatment decisions in advanced non-small cell lung cancer rely on accurate analysis of the EGFR mutation status in small tissue samples. Non-small cell lung cancer (NSCLC) accounts for more than 80 % of all lung cancers. About ten percent of NSCLC cancers harbor activating mutations in the Epidermal Growth Factor Receptor (EGFR) gene. The most common activating mutations of EGFR occur in the intracellular tyrosine kinase domain, i.e. in exons 18–21 [2, 3]. These mutations lead to constitutive activation and convey transforming capacity to the receptor. Deletions in exon 19 and a point mutation in exon 21 (L858R) account for about 90 % of EGFR
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