Snapback Primer Mediated Clamping PCR for Detection of EGFR and KRAS Mutations in NSCLC Patients by High Resolution Melting Analysis

Assays for detecting somatic mutations are requested with higher sensitivity and more convenience. Here, we describe snapback primer mediated allele clamping enrichment polymerase chain reaction (SPACE-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures. We replaced regular PCR with SPACE-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold in detecting EGFR and KRAS somatic mutations. Combined SPACE-PCR with analysis of snapback primer by high resolution melting (SPACE-HRM), the high sensitive system that enables a closed-tube detection of mutations after isolating mutants has been established, as low as 1/105–1/1000 mutant samples can be diagnosed. And finally, in a double-blind experiment of 150 cases of non-small-cell lung cancer (NSCLC) patients, compared with direct DNA sequencing and ADX-EGFR/KRAS mutation detection kit, up to 25% of the PCR-direct sequencing negative cases turned out to be positive in SPACE-HRM mutation tests; the specificity is 100%. Results demonstrated that the SPACE-HRM system we set up is a high sensitive assay that can be used for EGFR and KRAS allele enrichment and reliable detection. We anticipate that the method will be employed in multiple applications in the clinic, including diagnosis, scanner recurrence monitoring, and treatment management.


Introduction
Somatic mutations in the gene of epidermal growth factor receptor (EGFR) and KRAS are associated with sensitivity and resistance to the kinase inhibitors in targeted therapy. Detection of EGFR and KRAS mutations is now a necessary procedure for treatment of non-small-cell lung cancer (NSCLC) before using EGFR tyrosine kinase inhibitors (EGFR-TKI) [1][2][3][4]. For the heterogeneity in tumor tissues and dilution of wild type, the ratio of mutant to wild type DNA could be less than 10%. The methods must be sensitive enough to detect such low-frequency mutant samples.
Although direct DNA sequencing is often considered as a gold standard for the identification of mutations, it remains laborious and time consuming. Moreover, it might suffer from a lack of sensitivity about 10%-20% and robustness for the determination of mutations [5]. Alternative methods have therefore been developed to detect these common mutations based on restriction enzyme analysis, allele specific amplification, digital PCR, fluorogenic hybridization probes,  Figure 1: Snapback primer mediated clamping PCR and HRM analysis. We designed probe's sequence that matches wild type to make sure that the melting temperature of wild hairpin stem structure will be higher than mutant type. (a) HRM analysis by snapback primer genotyping after normal PCR. Tsp value is the middle melting temperature of wild and mutant snapback hairpin, which is the temperature chosen for extending in SPACE-PCR cycles. (b) The principle of SPACE-PCR. When extended in Tsp ( ∘ C), mutant can be enriched. (c) HRM analysis with snapback primer after SPACE-PCR (SPACE-HRM). After SPACE-PCR, both the melting curves of wild snapback hairpin and amplicon are reduced. Heterozygous samples intend to be pure mutant. and high resolution melting (HRM) [6][7][8][9]. HRM analysis is a post-PCR technique to identify genetic variation in nucleic acid sequences. It has been used to screen large numbers of potential variant sequences and genotype mutations in EGFR and KRAS detections [3,10,11]. As a one-step precursor to other more laborious techniques such as sequencing, it has incomparable superiority in clinical promotion. In 2008, Zhou et al. developed snapback primer into genotyping analysis by HRM without extra cost of special covalent modifications. After several thermal cycles of amplification, dependent on the amplicon sequence of snapback primer, a complementary probe portion of primer tail snaps back on its extension products so that an intramolecular hairpin is formed after the asymmetric PCR; then the melting curves of snapback hairpin were analyzed based on the difference of 3-6 ∘ C between wild and mutant genotyping [12].
In this study, we developed a snapback primer mediated allele clamping enrichment polymerase chain reaction (SPACE-PCR) for mutant-enrich amplification and HRM analysis in detecting EGFR and KRAS mutations. When in the cycling of amplification, the extending temperature is set between the wild and mutant melting temperature of hairpin stem structure to inhibit the amplification of wild template, while the mutant comes to be enriched successfully. Then, cooperated with analysis of snapback primer by high resolution melting (SPACE-HRM), mutant peaks must be more obvious, while wild peaks are weakened ( Figure 1). We developed SPACE-HRM assays for EGFR and KRAS common mutations that are currently used for drug sensitivity and resistance diagnoses. The enrichment efficiency of the SPACE-PCR was tested with sequencing and HRM analysis using a dilution series of mutant DNA into wild BioMed Research International In detection of EGFR E746-A750 del (15 bp), probe (22 bp) cannot cover the mutant site, so there is no peak of intramolecular snapback hairpins for mutant-type samples, while the peak value of wild type is 74.5 ∘ C. Besides this, difference also exists in peaks of intermolecular duplexes. When there is an obviously small peak 2 ∘ C below the main peaks of intermolecular duplexes, it means this sample just consists of wild sequence (peak a); then if 4 ∘ C-below, mutant exists (peaks b, c, and d). Mixed samples (peak b, 10% mutation, and peak c, 20% mutation) showed that the more mutant template the lower probe peak. In detection of KRAS, as for point mutation detection, wild-type (peak a) probe peak value is 74 ∘ C and about 5 ∘ C below is the mutation (peak d). Mixed samples (peak c, 50% mutation, and peak b, 20% mutation) showed that the higher the percentage, the higher the peak standing for.
DNA. Furthermore, we showed that false-negative results analyzed by method of direct DNA sequencing or Scorpion-ARMS could be identified by SPACE-HRM in a test of 150 cases of NSCLC patient samples.  After PCR, the products were denatured at 95 ∘ C (60 s hold), cooled to 55 ∘ C (60 s hold), and melted at 0.02 ∘ C/s with continuous acquisition of fluorescence until 95 ∘ C. After HRM analysis, the reaction volumes are delivered to DNA sequencing.

Melting Curves
Analysis. The melting of both intramolecular snapback hairpins and intermolecular amplicon duplexes is observed as peaks on negative first-derivative plots of fluorescence with respect to temperature by analysis method "Tm calling" that instrument LC480 II provides.

The Establishment of SPACE-HRM.
A characteristic of genotyping deletion and point mutant by snapback primer HRM after normal PCR is shown in Figure 2. For a large fragment of deletion (E746-A750 del (15 bp del)), probes we designed could not snap back with mutant to form a hairpin stem, with showing full-length amplicon melting only. For Then, the sensitivity of SPACE-HRM was compared with snapback primer HRM after normal PCR in Figure 3. Mutations accounted for 1, 1/10, 1/100, 5/1000, 1/1000, 1/10 4 , and 1/10 5 and wild samples were tested. Before enrichment, 1/10 mutant sample was the baseline. For deletions, it seems to be enriched more easily by SPACE-HRM. 1/10 5 mutant samples could be detected, while 1-bp mutation was improved to be 1/1000. Finally, to make sure of the enrichment efficiency, the enrichment results of SPACE-PCR were verified by DNA sequencing. Sequence diagrams of SPACE-PCR and normal PCR were compared in Figure 4. The sensitivity of direct sequencing was about 1/10. After mutant enrichment by SPACE-PCR, 1/10 4 deletion mutant-type and 1/200 point mutation have been analyzed.

Double Blind Testing of 150 Tissue Samples Compared with Sequencing and ADX-EGFR/KARS Mutation Detection Kit.
We extracted DNA, coded number 1-number 150, from 50paraffin tissue and 100 fresh tissue samples. Then, SPACE-HRM, direct DNA sequencing, and Scorpion-ARMS simultaneously diagnosed these samples with ADX-EGFR/KARS mutation detection kit (Amoy Diagnostics Co. Ltd., Xiamen, China). To compare the methods between SPACE-HRM, direct DNA sequencing, and Scorpion-ARMS with ADX-EGFR/ KARS mutation detection kit (Amoy Diagnostics Co. Ltd., Xiamen, China), DNA from 50-paraffin tissue and 100 fresh tissue samples coded from number 1 to number 150 were extracted and used to test simultaneously. 48 patients (49/150; 32%) were found to harbor activating EGFR and KRAS mutations using PCR-direct sequencing method, with a majority of patients (39/49; 80%) carrying exon 19 deletion or L858R point mutations. 101 cases negative for EGFR and KRAS mutations from PCR-direct sequencing were further analyzed using Scorpion-ARMS and SPACE-HRM technology. Up to 25% of the PCR-direct sequencing negative cases turned out to be positive in SPACE-HRM mutation tests, and this proportion is 20% by the Scorpion-ARMS. The amount of patients with different mutation by the three assays was in Table 1. Mutant details were listed in Supplementary Tables 2 and 3.

Discussions
Currently, DNA sequencing is also the "golden standard" of genotyping technologies, and Scorpion-ARMS is thought to be the most efficient method in detecting EGFR and KRAS mutations, which can be used to analyze mutations higher than 1%, and FDA US has accredited it in detection of EGFR and KRAS mutations. The specificity of SPACE-HRM is tested compared with these two standard methods. Results were convincing; the SPACE-HRM detection system might be a good choice for one-step mutation assay for its highest sensitivity.
A key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. For the purpose of obtaining high sensitivity, the mutantenrich PCR combined with high sensitive post-PCR analysis methods must be necessary for PCR-based assay. SPACE-PCR that enables exclusive amplification and isolation of the mutants would transform the capabilities of PCR-based genetic testing. It is operated in an easy way by changing extending temperature of thermal cycles. For mutation of large-fragment insertions and deletions, when probes cannot hybridize to the template, we can choose whatever temperature under the wild melting temperature of hairpin. For point mutations and small fragment mutants, temperature between mutant-type and wild-type hybridizing temperature is necessary. The difference is about 5 ∘ C, certainly closer to the wild and more efficient to mutant enrichment. But we often select the middle 2-3 ∘ C to make sure of the mutant-enrichment as well as the low-frequency mutant samples be amplification successfully. Compared with another enrichment method-COLD PCR, the temperature used for enrichment is easier to choose, which means the experiment's results can be more stable with being little affected by PCR instruments. Normal PCR thermal cycling instruments can well perform the method for mutant-enrich PCR. We have made a test in common PCR instruments with the same protocol described above, and the same results were obtained.
SPACE-PCR can be combined with any post-PCR analysis method, if sensitive enough, like HRM, digital PCR, and so on [13]. Ultrahigh sensitive assay will be developed. Here, we chose the snapback primer HRM and formed a simple and one-step platform for clinical use.
Nowadays, many high sensitive technologies were developed to detect circulating cell-free DNA (cfDNA) in plasma or serum [11,[14][15][16], which is useful for numerous diagnostic applications and would avoid the need for tumor tissue biopsies. Since large difference of cfDNA concentration and integrity exists in different samples, methods with cluster analysis that need a uniform concentration of starting template seem to be not ideal, such as PNA/LNA-mediated realtime PCR clamping, TaqMan probe ARMS, and Scorpion-ARMS primers for SNP real-time PCR detection [1,[17][18][19]. We distinguish wild and mutant by the shape of the melting peaks instead of the amplification Ct values, which means the concentration homogenization of DNA before reaction and the cluster analysis are not needed. A small amount sample of pretest has been done in cfDNA detection, and results were consistent with what we expected. Besides, it can be better for use, when samples are not fresh or are extracted with the different methods. Precisely because of its high sensitive and intuitive way of distinction without comparisons of references, our high sensitive SPACE-HRM system may be a good choice for circulating peripheral blood analysis. The experiment in this area continues to be done.

Conclusions
We developed a new method for mutant-enrich PCR only with snapback primer (SPACE-PCR), in which the enrichment efficiency is nearly 100-fold. The efficiency is particularly obvious for large fragment mutations. With this method, we can detect lower than 1/10 mutant samples by sequencing. Then, new snapback primer HRM assays with innovative SPACE-PCR technology (SPACE-HRM) were specifically developed to detect somatic EGFR and KRAS mutations that are currently used for drug sensitivity and resistance diagnoses. By combining the power of the two technologies, a simple, fast, highly sensitive, and high throughput DNA detection system for one-step detection of clinic application was established, with which we can detect 1/1000-1/10000 low-mutant samples. In large fragment mutations, like EGFR