Validation of a locked nucleic acid based wild-type blocking PCR for the detection of EGFR exon 18/19 mutations

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

Ground-glass opacity heralding invasive lung adenocarcinoma with prodromal dermatomyositis: a case report

“…While previous studies have documented the association between histologic types of lung cancer and DM, little attention has been paid to its association with the molecular basis of malignancy. Genetic analysis of the primary tumor in the present case revealed mutation of the gene encoding EGFR, an event reported to occur in about 10% of cases of non-small cell lung cancer [ 54 ]. Point mutations of the codon for G719 account for about 3% of all EGFR mutations in lung cancer; are more common among non-smokers, females, and East Asians [ 55 ]; and predict sensitivity to EGFR tyrosine kinase inhibitors [ 56 ].…”

Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation

“…Because of this, LNAs have been used in multiple applications [42][43][44]. Interestingly, LNA oligonucleotides have been used to block the PCR amplification of unspliced transcripts (by targeting the intronic sequence) [45] or wildtype transcripts when the mutated version is of interest [46][47][48]. A patent describing the use of LNA oligonucleotides to block reverse transcription and amplification of hemoglobin mRNA from whole blood during RT-qPCR [49] further exemplifies their potential and applicability for depletion purposes.…”

Blocking abundant RNA transcripts by high-affinity oligonucleotides during transcriptome library preparation

“…Because of this, LNAs have been used in multiple applications (Breitenbuecher et al, 2014; Singh et al, 1998; Zhang et al, 2012). Interestingly, LNA oligonucleotides have been used to block the PCR amplification of unspliced transcripts (by targeting the intronic sequence) (Hummelshoj et al, 2005) or wild-type transcripts when the mutated version is of interest (Dominguez and Kolodney, 2005; Oldenburg et al, 2008; Vliegen et al, 2015). A patent describing the use of LNA oligonucleotides to block reverse transcription and amplification of hemoglobin mRNA from whole blood during RT-qPCR (Russell et al, 2006) further exemplifies their potential and applicability for depletion purposes.…”