A novel method for detection of mutation in epidermal growth factor receptor
Introduction
Lung cancer is one of the leading causes of cancer-related death in people and non-small cell lung cancer (NSCLC) accounts for 80–85% of cases. EGFR mutations in NSCLC have been reported sensitivity to the tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) or erlotinib (OSI-774, Tarceva) by many authors [1], [2]. Subsequently, the correlation between EGFR mutations and EGFR-TKIs was extensively investigated [3], [4], [5], [6], [7], [8], [9]. The two hot-spot mutations consist of the 15-bp nucleotide in-frame deletion in exon 19 (E746_A750del) and the point mutation replacing leucine with argentine at codon 858 in exon 21 (L858R), which account for approximate 90% of all EGFR mutations [8], [10], [11].
Because of the strong association with clinical treatment response, screening of EGFR hot-spot mutations prior to selection for therapeutic strategy has been considered extremely valuable. However, determining mutation status of EGFR is challenging. The major difficulty is that EGFR mutant cells are mixed by a large amount of wild cells derived from the site of tissue sampling. Currently, many of methods to overcome this obstacle have been reported, including PCR–RFLP analysis [12], [13], co-amplification at lower denature-PCR [14], suspension array [15], ARMS PCR [16], [17], [18], [19]. Among them, mutant-enriched PCR and ARMS PCR have been the major means for mutant alleles genotyping. The mutant-enriched PCR can selectively cut wild type alleles and leave the mutant alleles enriched. This approach has been successfully applied in the detection of p53, Ras and EGFR mutation [13], [20], [21], [22]. ARMS PCR is based on the principle that extension is efficient when the 3′ terminal base of a primer matches its target, whereas extension is inefficient or nonexistent when the 3′ terminal base is mismatched. This strategy has been also successfully performed on the screening of point mutations [23], [24], [25]. However, mutant-enriched PCR involves many steps of post-PCR processing which may increase PCR product contamination. ARMS method is also confronted with infraction amplification by wild allele, lacking of sensitivity of detection.
As we know, nobody has combined mutant-enriched PCR with ARMS real-time PCR. Herein, we report a novel method that mutant-enriched PCR and ARMS TaqMan real-time PCR are coupled in a one-step reaction tube. This method has been used to screen the EGFR hot-spot mutations (E746_A750del and L858R) in 73 clinical samples.
Section snippets
Plasmid construction
Recombinants containing EGFR wild types of exon 19 and exon 21, and mutant types of E746_A750del in exon 19 and L858R in exon 21, were constructed according to a method reported by Board et al. [25]. Briefly, corresponding outer and mutant primers were used to yield half fragments with complimentary ends, each half fragment containing a mutant base. These PCR products were mixed and amplified with inner nest primers. Self-priming of the complementary half fragments and subsequent amplification
Cut-off value determination
To determinate the cut-off value of the ARMS method, we performed ARMS Taqman PCR and internal control Taqman PCR using 10 wild genomic DNA samples with concentration ranging from 5 ng/reaction to 50 ng/reaction. The assays were performed 5 times and each concentration was repeated in triplicate in each assay. The cutoff ΔCt value was determined to be 3 Ct below the lowest ΔCt value observed in all reactions for each assay. The cut-off ΔCt values of E746_A750del and L858R were both 12.
Sensitivity
To
Discussion
For mutation detection, direct sequencing is one of the most common methods. Sequencing is a straightforward method and is widely used [27], [28], [29]. However, the detection limit for direct sequencing is generally about 25–30% mutant alleles of total genomic DNA content [12], [30]. Results of our mutant-enriched PCR sequencing method show it is more sensitive than the direct PCR sequencing, which is able to detect less than 10% mutant alleles of total genomic DNA content. It confirms that it
Conflict of interest statement
All authors declared that they had no any conflicting interest.
Acknowledgments
The project is supported by Special Foundation of President of the Chinese Academy of Sciences and the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP) and Research Fund for Middle-aged and Young Scientist (2010C7) and Infectious Diseases Special Project, Minister of Health of China (2008ZX10003—009).
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These authors contributed equally to this work.