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J Mol Diagn. Sep 2011; 13(5): 485–492.
PMCID: PMC3157611

A Multiplex SNaPshot Assay as a Rapid Method for Detecting KRAS and BRAF Mutations in Advanced Colorectal Cancers

Abstract

The analysis of KRAS mutations has become a prerequisite for anti-epidermal growth factor receptor therapy in patients with metastatic colorectal cancers. KRAS mutations are associated with resistance to treatment by monoclonal antibodies such as cetuximab and panitumumab and thus are correlated with a shorter progression-free survival. BRAF mutations also may play a role in treatment decisions. The widespread use of these targeted therapies has generated the need to develop cost-effective methods for routine KRAS and BRAF analysis. The aim of this study was to compare a multiplex SNaPshot assay with DNA sequencing and high-resolution melting analysis for identifying KRAS codons 12 and 13 and BRAF codon 600 mutations. Thus 110 routinely formalin-fixed and paraffin-embedded tissue blocks were tested by each method. The SNaPshot analysis detected KRAS and BRAF codon 600 mutations in, respectively, 34.5% (n = 38) and 10% (n = 11) of these tissue blocks. These results were confirmed by direct DNA sequencing and by high-resolution melting analysis. The costs and time constraints of each detection method were compared at the same time. In conclusion, our newly designed multiplex SNaPshot assay is a fast, inexpensive, sensitive, and robust technique for molecular diagnostic practices and patient selection.

Mitogen-activated protein kinase and phosphatidylinositol-3′-OH kinase signaling pathways form a network that participate in tumorigenesis. Activation by mutation of the different adaptors of this molecular network will deregulate proliferation, differentiation, and cell survival.1 In colorectal cancers (CRCs), the KRAS, BRAF, and PIK3CA genes are mutated in approximately 30% to 50% of cases, leading to the activation of Ras/Raf/mitogen-activated protein kinase and phosphatidylinositol-3′-OH kinase signaling pathways.2,3 Targeting epidermal growth factor receptor (EGFR) with monoclonal antibodies such as cetuximab and panitumumab has been shown to be an effective therapy for metastatic colorectal cancer as a single agent or in combination with chemotherapy. Activation of mutations in codons 12 and 13 of KRAS is associated with resistance to both cetuximab and panitumumab in patients with metastatic CRC.4,5 On the basis of these results, the American Society of Clinical Oncology and the National Comprehensive Cancer Network both recommend KRAS mutation testing before prescribing EGFR antagonist therapy for patients with metastatic CRC and have stated that alternative therapies should be prescribed when mutations are detected.4,6,7 Nevertheless, tumors with wild-type KRAS account for approximately 30% to 40% of resistant patients. Recent findings suggest that as with KRAS, mutation of the BRAF gene involving codon 600 is associated with a low overall response rate to cetuximab and panitumunab and a poor prognosis.8,9 Today KRAS and BRAF status are considered in selected patients with metastatic CRC who are candidates for anti-EGFR therapy. Recent studies also have demonstrated that in addition to KRAS and BRAF, mutations in PIK3CA exon 20 were associated with a low response rate to EGFR inhibitors in patients with wild-type KRAS tumor status.10,11 However, because of the low numbers of patients with PIK3CA exon 20 mutations in the series, the size of the cetuximab resistance effect is uncertain, and these data need to be confirmed.

In this study we developed and validated in routine diagnostics a sensitive SNaPshot test that allows for the rapid and simultaneous identification of common hot-spot mutations in KRAS and BRAF oncogenes in persons with advanced CRC. Various methods have been described for the detection of KRAS and BRAF mutations such as pyrosequencing,12,13 real-time PCR,14 array analysis,15 amplification refractory mutation system (Scorpion assay)16, high-resolution melting (HRM) analysis,15–18 and Sanger sequencing of PCR products.9,15–18 The disadvantage of DNA sequencing is its relatively high cost and limited sensitivity. HRM analysis is a recently developed molecular technique that is a cost-efficient, closed-tube system without any post-PCR processing and shows greater sensitivity and specificity than Sanger sequencing for KRAS and BRAF mutation detection.17,19 A primer extension-based method, SNaPshot, has been described previously for KRAS codons 12 and 13 mutations.20 This method may be adapted for the simultaneous analysis of up to 50 biallelic single nucleotide polymorphisms (SNPs) in a single reaction. We and others have developed SNaPshot assays that are able to detect KRAS and BRAF mutations simultaneously on routinely used formalin-fixed and paraffin-embedded (FFPE) blocks.21–23 However, currently it is very difficult to tell which test is the most reliable. Moreover, tumor samples are very heterogeneous with regard to fixation, biopsy, surgical specimen, and neoadjuvant treatment, and one “best” method could depend on the type of sample.

We report herein our experiment (validated in every experiment performed during daily routine) in screening for KRAS codons 12 and 13 and BRAF codon 600 by SNaPshot, HRM analysis, and sequencing. Because it is essential to screen patient samples rapidly and at little expense, the cost-effectiveness and the total necessary time to acquire results were compared for each of the three methods used.

Materials and Methods

Samples and DNA Extraction

FFPE tissue samples were obtained from 110 consecutive patients with advanced CRC who were treated in the Cancer Institute of Franche-Comte, France. Informed consent was obtained from all patients. FFPE tissues containing at least 60% tumor cells were selected after microscopic examination by a pathologist. For 42 samples, ten 10-μm-thick serial sections were cut from paraffin blocks and placed in a microcentrifuge tube for DNA extraction. For the other 68 paraffin blocks that contained <60% of tumor cells, a coring procedure was used to enrich for tumor cell DNA. DNA extraction was performed with use of the QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France) according to the protocol for fixed tissues. DNA was eluted twice in a volume of 50 μL and then quantified spectrophotometrically at 260 nm with a Nanodrop ND-1000 (Labtech, Palaiseau, France). All DNA samples were tested for integrity and amplifiability using four pairs of primers for Tbxas exon 9, Rag1 exon 2, Plzf exon 2, and Af4 exon 21 amplification in a multiplex PCR to generate products with a length of 100, 200, 300, and 400 bp (BIOMED-2 control gene PCR method).24 Because the quantity of DNA does not reflect the template quality, the multiplex PCR control was conducted on all extracted DNA. PCR was performed in a 25 μL reaction containing 1× Qiagen Multiplex PCR Master Mix (Qiagen), Plzf and Af4 primers at 0.5 μmol/L final concentration, Tbxas and Rag1 primers at a final concentration of 0.25 μmol/L, and at least 3 ng template DNA (2.5 μL volume of extracted DNA). The following protocol was used: 95°C for 10 minutes, 34 cycles of 95°C for 45 seconds, 60°C for 1 minute, 72°C for 1 minute 30 seconds, and finally 10 minutes at 72°C. PCR products were controlled by running 15 μL in 2% agarose–Tris-borate-EDTA gel.

Plasmid Standards

Plasmids containing each of the seven most frequent mutations of KRAS (p.G12V, p.G12A, p.G12D, p.G12S, p.G12C, p.G12R, and p.G13D) and the wild-type KRAS exon 2 were prepared. PCR amplification of the target genes was performed using genomic DNA extracted from tumor cells of FFPE tissue samples and that had been previously sequenced and shown to carry the KRAS and BRAF mutations. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and cloned into a pGEM-T Easy vector using a TA Cloning Kit (Promega, Charbonnières-les-Bains, France) according to the manufacturer's instructions. Colonies containing recombinant plasmids were picked and sequenced to validate recombinant plasmids. Plasmids were quantified spectrophotometrically. A dilution series for each plasmid carrying the mutant allele was prepared to evaluate the sensitivity of the SNaPshot assays. The dilution series consisted of 0%, 1%, 5%, and 10% of mutant plasmids diluted into wild-type plasmid. Cloned DNA encoding each mutation analyzed in this work and required to control the SNaPshot multiplex assay will be available on request.

KRAS Exon 2 and BRAF Exon 15 Multiplex PCR Amplification

A multiplex PCR assay was designed to amplify fragments of KRAS exon 2 (200 bp) and BRAF exon 15 (250 bp). Primers for multiplex PCR amplification were as follows: KRAS exon 2, 5′-AAGGCCTGCTGAAAATGACTG-3′ (forward), 5′-CAAAGAATGGTCCTGCACCAG-3′ (reverse); BRAF exon 15, 5′-CATAATGCTTGCTCTGATAGGAAA-3′ (forward), 5′-TCAGCAGCATCTCAGGGCCAAA-3′ (reverse). Multiplex PCR was performed with the Qiagen Multiplex PCR Kit (Qiagen) in a total volume of 25 μL containing 1× Qiagen Multiplex PCR Master Mix (providing a final concentration of 3 mmol/L MgCl2), 0.5 μmol/L of each primer, and DNA extracted from paraffin-embedded material (2.5 μL containing at least 3 ng of DNA) or 2.5 μL of sterile water as a negative control. PCR conditions were: 94°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 58°C for 1 minute, 72°C for 1 minute, and finally, 10 minutes at 72°C. Multiplex PCR products were checked for quality and yield by running 18 μL in 2% agarose–Tris-borate-EDTA gel. In case no amplified band was detected, a second run of PCR was made to increase the detection threshold. Five microliters of primary amplification product was used as a template for the secondary amplification with the conditions previously described, except that the number of cycles was limited to 25. The secondary multiplex PCR assay was applied to the previous negative control to detect possible contaminant amplification. PCR products from the second amplification were separated by electrophoresis on 2% agarose gels, stained with ethidium bromide, and visualized on a UV light transilluminator. After purification using the gel extraction kit NucleoSpin Extract II (Macherey-Nagel, Hoerdt, France), PCR products were analyzed for the presence of KRAS codon 12 and codon 13 mutations and BRAF codon 600 mutational status. Separate isolation of both DNA fragments using agarose gel is not necessary.

SNaPshot Analysis

SNaPshot analysis was performed prospectively for diagnostic purposes with use of the ABI Prism SNaPshot Multiplex kit (Applied Biosystems, Courtaboeuf, France). Amplified KRAS exon 2 and BRAF exon 15 were analyzed for the presence of KRAS mutations at nucleotides c.34, c.35, c.37, and c.38 and for BRAF mutation at nucleotide c.1799 using five primers that contained an additional poly(dC) tail at their 5′ end, allowing for their simultaneous detection (Table 1). Primers used for KRAS codons 12 and 13 were previously described.20 Reactions were performed in a final volume of 5 μL, containing 1.5 μL of purified multiplex PCR product (2 to 10 μg/μL), 2.5 μL of SNaPshot Ready Multiplex Ready Reaction Mix, 0.5 μL of probe equimolar mix (each probe at 2 μmol/L final), and 0.5 μL of double-distilled water. Multiplex single base extensions were carried out for 25 cycles according to the following program: 10 seconds at 96°C, 5 seconds at 50°C, and 30 seconds at 60°C. SNaPshot products were then treated at 37°C for 1 hour with 1 μL of shrimp alkaline phosphatase at 1 U/μL diluted in 2.5 μL of shrimp alkaline phosphatase buffer 10× and 11.5 μL of double-distilled water. After heat inactivation of shrimp alkaline phosphatase for 15 minutes at 75°C, 2 μL of the labeled products were mixed with 9.5 μL of HiDi formamide and 0.5 μL of Genescan-120LIZ size standard. They then were separated using a 25 minutes run on an ABI Prism 3130 DNA sequencer with POP-7 matrix and 14 seconds for injection. The analysis was performed using GeneMapper ID software version 3.2.1 (Applied Biosystems).

Table 1
SNaPshot Primers for the Detection of KRAS Codons 12, 13, and BRAF Codon 600 Mutations

Sequencing Analysis

To confirm SNaPshot results, the purified multiplex PCR products were sequenced for the detection of KRAS mutations in exon 2. The sequencing reaction was performed using the Big Dye terminator V3.1 cycle sequencing kit (Applied Biosystems) and the same reverse primer used for the multiplex PCR. The reaction mix consisted of 1 μL of terminator premix 1×, 0.5 μL of sequencing buffer 5×, 1 μL of primer at 10 μmol/L, 2.5 μL of double-distilled water, and 5 μL of cleaned template (25 to 50 ng/μL) in a total volume of 10 μL. The reactions were run according to the following protocol: one cycle of 96°C for 1 minute; 15 cycles of 96°C for 10 seconds, 50°C for 5 seconds, 60°C for 1 minute 15 seconds; five cycles of 96°C for 10 seconds, 50°C for 5 seconds, 60°C for 1 minute 30 seconds; five cycles of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 2 minutes. After purification with a NucleoSEQ kit (Macherey-Nagel), samples were run on an ABI Prism 3130 DNA sequencer and analyzed using Sequencing Analysis software version 5.2 (Applied Biosystems).

HRM Analysis

An HRM assay also was performed to detect KRAS and BRAF mutations using the same DNA preparation as for the multiplex PCR. Real-time PCR and HRM analysis were carried out consecutively on a LightCycler 480 (Roche Diagnostics, Meylan, France). PCR for KRAS exon 2 was performed as previously reported.25 For BRAF, primers were selected to flank the V600E mutation in exon 15 and were designed as follows: 5′-ATAGGTGATTTTGGTCTAGCTACAG-3′ (forward), 5′- AGTAACTCAGCAGCATCTCAGG-3′ (reverse). Each reaction mixture contained 10 μL of LightCycler 480 High Resolution Melting Master 2× (Roche Diagnostics), 3 mmol/L of MgCl2, 200 nmol/L of each primer, 0.5 U of Uracil-DNA Glycosylase, 1.1 μL of double-distilled water, and 5 μL (1 to 100 ng) of genomic DNA. Incubation of the reactions with uracil-DNA glycosylase for 10 minutes at 40°C before PCR prevents carryover contamination. PCR conditions were: 95°C for 10 minutes, followed by 50 cycles of 10 seconds at 95°C, a touchdown of 63°C to 58°C for 10 seconds (1°C/cycle), and 10 seconds at 72°C. After amplification, the PCR product was denaturated at 95°C for 1 minute and cooled down to 40°C for 1 minute to allow for heteroduplex formation. The final step was performed from 70°C to 95°C with an increase of 1°C per second with 25 acquisitions per degree. The HRM curve analysis was performed with use of Gene Scanning software (Roche Diagnostics). All samples were run in triplicate.

Statistical Analysis

Results of KRAS and BRAF mutational analyses were used as categorical variables (presence or absence of the mutation). The correlation of KRAS mutation frequency with sections of FFPE tissue punch cores was assessed by Fisher's exact test. Statistical significance was set at P < 0.005.

Results

DNA Fragmentation Analysis

For any PCR-based technique, template quality is probably the most important factor. Before the analysis of the KRAS and BRAF mutational status, DNA quality control was performed using a PCR for four different-sized fragments (100, 200, 300, and 400 bp in length) of reference genes. The FFPE samples produced variable numbers of bands reflecting variable DNA integrity. Among the 110 DNA samples obtained from FFPE samples, 72 produced bands of 100 to 400 bp and 15 allowed amplification of 100 to 300 bp. For nine samples, PCR products of 100 to 200 bp could be amplified, and finally, 14 samples allowed amplification only of the smallest sized control amplicon of 100 bp.

Multiplex PCR and SNaPshot Analysis

The first development made to the SNaPshot assay was to optimize a single-tube multiplex PCR able to amplify both targets of interest simultaneously from the FFPE samples in a routine diagnostic situation. Small PCR product sizes (200 bp for KRAS exon 2 and 250 bp for BRAF exon 15) were chosen to optimize amplification of partially degraded genomic DNA obtained from FFPE samples. The minimal amount of template to generate sufficient PCR products for SNaPshot or DNA sequencing was estimated at 3 ng and thus was not a limiting factor. To detect the KRAS 200 bp fragment and the BRAF 250 bp fragment, 32 of the 110 tumor samples (29%) with only 100 to 200 bp and very faint 200 to 300 bp signals after quality control PCR needed a second amplification of the first PCR product. To prevent the risk of nonspecific amplification, we limited the number of cycles in the second PCR assay (20 cycles), and negative controls from the first reaction underwent the second run. With use of these conditions, no contamination of the negative controls was observed.

PCR products were used as a template to perform a SNaPshot assay based on a single-base extension method and were able to detect simultaneously four different SNPs of KRAS exon 2 and the c.1799T>A BRAF exon 15 mutation (p.V600E). Somatic mutations of KRAS occurring at bases c.34, c.35, c.37, and c.38 from the start codon give rise to amino acid substitutions at codon 12 and 13. In this method, the primers described in Table 1 anneal one base 5′ of the locus that is to be typed. The DNA polymerase contained in the reaction mix appends a single fluorescently labeled dideoxynucleotide triphosphate to the 3′ end of the DNA primer. Detection of extended products was based on the four different fluorescently labeled dideoxynucleotide triphosphates and extension primers with oligonucleotide tails of differing lengths, thus controlling the concise length of the entire chromatogram to 60 bases. In our experiment, the difference in length between adjacent primers is 10 bp. Mutations are easily identified on the basis of peak size and color. When a mutation is present, an alternative dideoxynucleotide triphosphate is incorporated, resulting in a different colored peak. A mutant peak is considered positive if it is three times above background noise in the wild-type sample. The mobility of extended primers in capillary electrophoresis is determined by their size, nucleotide composition, and dye.

Validation and Sensitivity of the SNaPshot Assay

Validation was performed using primary tumor samples that had been previously tested by sequencing and shown to carry the mutations of interest. A representative example of common mutations detected is shown in Figure 1. In the wild-type sample electropherogram (Figure 1A), the four blue peaks indicate the four known SNPs of KRAS gene codons 12 and 13, and the red peak corresponds to the codon 600 SNP of the BRAF gene. In mutant samples, an additional peak overlapping or juxtaposing one peak of the wild-type electropherogram is indicative of the presence of a heterozygous mutation. In Figure 1B, two peaks at nucleotide c.34 showing “G” and “C” genotype indicate a c.34 G>C heterozygous mutation resulting in a p.G12R KRAS protein mutation. Figure 1, C–E, shows a c.35 G>A; p.G12D, a c.37G>T; p.G13C, and a c.38G>A; p.G13D KRAS mutation, respectively.

Figure 1
Assay validation of SNaPshot genotyping. Each peak corresponds to a specific extended primer. Positions of nucleotides are indicated at the bottom of the figure. Arrows indicate the location of each mutation. Bases are represented by the following colors: ...

To evaluate the sensitivity of SNaPshot assays, DNA from mutant plasmids harboring mutations of interest was diluted into wild-type plasmid in proportions of 10%, 5%, and 1%. Our experiments on plasmid model systems indicated the possibility of identifying at least 5% of mutated alleles in a background of wild-type DNA. Examples of sensitivity data are illustrated in Supplemental Figures S1 to S7 (available at http://jmd.amjpathol.org).

Mutational Analysis of KRAS and BRAF Genes in Clinical Samples

We used multiplex PCR plus SNaPshot to test 110 FFPE colon tumor samples collected routinely. KRAS mutations were found in 38 (34.5%) of 110 tumor samples analyzed, and BRAF c.1799 T>A; p.V600E mutation was detected in 11 patients (10%). Of the 38 KRAS mutant cases, 32 cases (29%) of codon 12 mutation were found, including 15 cases (13.6%) of p.G12D, seven cases (6.4%) of p.G12V, five cases (4.5%) of p.G12A, four cases (3.6%) of p.G12C, and one case (0.9%) of p.G12S. Six cases (5.5%) of codon 13 mutation were found, as were all of the p.G13D mutant (Table 2). This finding is in line with the frequencies reported in the literature2,3 and is consistent with previously published reports showing that KRAS codons 12 and 13 and BRAF codon 600 mutations are mutually exclusive.8,19 The c.37G>T; p.G13C and c.37G>C; p.G13R mutations in CRCs are rare (0.8% and 0.3% respectively). Probably because of the low frequency, no mutation was detected by the KRAS c.37 assay in our cohort. Nevertheless, we validated before the c.37 mutation detection using a lung adenocarcinoma sample that had been previously tested and shown to carry the KRAS c.37G>T mutation (Figure 1). Tumor sample extraction from paraffin-embedded tissue is another variable parameter. We then attempted to find a correlation between the detection of mutations in the KRAS gene and specimens prepared either by sections or cores in paraffin blocks. KRAS was mutated in 25 (37%) of 68 coring samples and in 13 (31%) of 42 sections (Table 3). No significant correlation was noted between the frequency of KRAS mutation detection and the specimens preparation (P = 0.42, Fisher's exact test).

Table 2
Distribution of Detected KRAS Mutations as Determined by SNaPshot
Table 3
KRAS Mutations Detection Frequency in DNA Extracted from Tissue Sections and Cores in Paraffin Blocks

Comparison of Molecular Testing Methods

We compared the multiplex SNaPshot assay with DNA sequencing and HRM analysis. All diluted plasmid mixtures used for SNaPshot sensitivity assessment were tested by HRM analysis and sequenced. Sequencing was sensitive to 5% to 10%, whereas HRM analysis readily detected 5% mutant sequence (data not shown). We next compared the results obtained by SNaPshot with those obtained by BigDye Terminator sequencing on the 110 FFPE colon tumor samples (Figure 2). The mutational analyses of KRAS codons 12 and 13 were successfully performed in 100% of cases and confirmed using both methods. In HRM assays, all of the DNA samples were amplified and analyzed for KRAS exon 2 and BRAF exon 15. Because KRAS and BRAF mutations are predominantly mutually exclusive, with very few tumors (0.6%) containing a mutation in both genes,9 it seemed reasonable to test for the presence of BRAF mutation by using the HRM assay only in wild-type KRAS tumors (72 samples) (Figure 2). All cases showing KRAS or BRAF mutations by SNaPshot were positive in HRM analysis, and no supplementary mutation was identified. HRM for KRAS mutations was performed using primers designed to span the entire exon 2 with a product size of 170 bp as reported.25 Samples that had been previously tested and carried the KRAS mutations were included for comparison. Examples of melting curves and difference plots of the HRM data obtained from the FFPE colon tumor samples are shown in Figure 3A. For BRAF mutations analysis, a shorter 151 bp PCR amplicon with one single melting domain was designed to have a better resolution of genotypes and increase the sensitivity of mutation detection.17 A wild-type control (DNA from the wild-type cell line) was used to normalize melting profiles of the other samples. The HT29 cell line, which carries a heterozygous c.1799T>A; p.V600E mutation, was used as a positive control (Figure 3B). Because any DNA alteration may produce an abnormal melting point curve, abnormal curves must be confirmed by an additional experiment, such as allele-specific competitive blocker PCR, direct sequencing, or pyrosequencing, which also show mutation identity.25–27 In our experiment, each BRAF mutation was confirmed by direct sequencing.

Figure 2
Strategies used to compare time and cost per sample for the three methods used to detect KRAS and BRAF mutations. Strategy 1: The 110 DNA samples were submitted to SNaPshot after KRAS exon 2 and BRAF exon 15 amplification detecting KRAS and BRAF mutations ...
Figure 3
High-resolution melting analysis of BRAF exon 15 and KRAS exon 2. A: Difference plots of one colorectal cancer sample carrying the p.V600E mutation. BRAF mutated sample was normalized against wild-type DNA. Products of the positive control (HT29 cell ...

Laboratory Working Time and Costs

The time and cost of each of the three methods used here for identifying KRAS codons 12 and 13 and BRAF codon 600 mutations (five SNPs) on each sample also were evaluated and reported in Figure 2. All procedures start with extracted DNA. Total time required was established at 6 hours 20 minutes for SNaPshot analysis versus 5 hours 30 minutes for automatic sequencing in an ABI Prism 3130 and 4 hours 10 minutes for HRM analysis in LightCycler480. When five SNPs are analyzed, SNaPshot using the ABI Prism SNaPshot Multiplex Kit costs 8.5€ (~$11.50 U.S.) per sample. This cost could be further reduced when more SNPs are simultaneously analyzed. In comparison, sequencing reaction using the Big Dye terminator V3.1 cycle sequencing kit costs 20€ (~$27 U.S.) per sample. DNA sequencing detection of KRAS and BRAF mutations involves KRAS exon 2 and BRAF exon 15 sequencing in two separate reactions, which increases the cost per sample. The HRM methodology is inexpensive [8€ (~$11 U.S.)], but the need for confirmation with a second method, such as sequencing, increases turnaround time and cost.

Discussion

Because only the predictive values of KRAS codons 12 and 13 and BRAF codon 600 mutations have been confirmed clinically in patients with CRC treated with anti-EGFR antibodies,11,25 we developed a SNaPshot assay as a diagnostically useful screening method for these common mutations in clinical FFPE specimens. The SNaPshot was validated in 110 routine heterogeneous tissue sets. Indeed, paraffin blocks are highly heterogeneous with respect to the quantity and distribution of tumor cells within the blocks, which is why a coring procedure was sometimes used to enrich for tumor DNA (always >60% of tumor cells), although no clear relationship was noted between the detection of mutation and the percentage of tumor sections.16 According to pathology laboratory practices in the Franche-Comte region, particularly formalin fixation, the genomic DNA isolated from FFPE tissue is more or less fragmented, as DNA quality control PCR showed. Multiplex PCR developed before SNaPshot step amplifies small DNA fragments (200 bp for KRAS and 250 bp for BRAF). For poor-quality FFPE samples (with degraded or low-yield DNA), a second round of PCR was undertaken to amplify targets and increase the overall yield of the amplified fragments. This strategy allowed all of the 110 FFPE samples to be tested.

The multiplex SNaPshot assay described here is able to examine simultaneously four nucleotides in KRAS exon 2 and one nucleotide in BRAF exon 15 for 12 possible point mutations for KRAS and one for the BRAF gene. According to the Catalogue of Somatic Mutations in Cancer, for CRC, ~100% of the mutations in BRAF codon 600 are p.V600E. Typically the p.V600E BRAF protein mutation is a result of the T>A mutation at position 1799 of the BRAF nucleotide sequence. This p.V600E mutation also can result from the two-base mutation TG>AA at nucleotide positions 1799 to 1800. In this study, samples evaluated for activating mutations of BRAF underwent direct DNA sequencing. All p.V600E mutations resulted from a single bp change of T to A at nucleotide position 1799. Other mutations also can occur at BRAF codon 600, especially in melanoma.18 These include p.V600K, p.V600D, and p.V600R. Here, the single base extension primer used for BRAF analysis at position 1799 also could detect a p.V600D mutation (c.1799_1800TG>AT). A confirmation of the p.V600 mutation is then necessary by using a reverse extension primer.

A variety of methods have been applied for somatic analysis of KRAS and BRAF mutations. Our results indicate that all methods used in this study—SNaPshot, direct sequencing, and HRM—yield similar results.

As with SNaPshot, direct Sanger sequencing is able to identify the specific mutation that may be present. However, to be successful, this methodology needs a sufficient amount of high-purity tumor material, probably because of the low sensitivity of the method previously reported.26 Indeed, it is known that the quality of FFPE-derived DNA is not always good for PCR or sequencing reactions, and this was indeed the reason for the failed samples. We previously made this point with an assay performed in 160 tumor samples, which showed 35 mutations and 125 wild-type KRAS. In this previous cohort of 160 samples, SNaPshot analysis was able to identify KRAS mutations in 11 cases originally considered wild type, using direct sequencing analysis (data not shown). For this previous cohort, the percentage of tumor sections was not determined so far, and results were directly related to the poor DNA quality and to the reported difference in sensitivity between the two methods (~20% for dideoxy sequencing and 5% for SNaPshot).23,26 In the assay reported here, the high concordance of results assessed by sequencing and SNaPshot most likely reflects the use of DNA extracted from samples consisting of at least 60% tumor cells and the improvement of fixation procedures.

Because a sensitive method based on HRM analysis to characterize KRAS and BRAF mutations has been described recently, we compared HRM with SNaPshot.17,19 As published previously, HRM represents a more sensitive approach than direct DNA sequencing to detect somatic mutations in tumoral tissue with a sensitivity similar (5% to 6%) to that observed with SNaPshot25 Moreover, HRM seems to be a suitable, fast, closed-tube methodology for testing FFPE samples.16,17,19,28 This screening methodology also has the advantage of using a very low quantity of DNA. Our experiment revealed that about 1 ng of genomic DNA isolated from an FFPE sample is sufficient to obtain a result directly, whereas a second amplification sometimes is required to obtain a PCR product for a SNaPshot reaction. This observation is probably explained by the fact that the first step of the HRM assay is a simplex PCR reaction using LC Green for increasing the sensitivity of DNA detection. Moreover, HRM products (151 bp for BRAF exon 15 and 170 bp for KRAS exon 2) are shorter than SNaPshot multiplex PCR products (250 bp for BRAF and 200 bp for KRAS). However, despite these advantages, HRM analysis does not provide the identity of mutations that are potentially detected and required another method to confirm the mutation.

In conclusion, we have developed and validated a multiplex SNaPshot methodology for the robust and reliable genotyping of KRAS and BRAF mutations in a fast, simple, and cost-effective manner. Moreover, we provide a procedure for amplifying low-quality DNA (eg, DNA that is degraded or overfixed) by using two rounds of PCR and allowing for the analysis of KRAS and BRAF mutation in such samples. Because SNaPshot can analyze more than 10 SNPs at the same time in a single reaction, it is not a closed method. It therefore could allow for the simultaneous routine detection of common mutated oncogenes (ie, for KRAS codon 61) and thus the eligibility of patients with metastatic CRC for anti-EGFR therapy.

Acknowledgments

We thank Drs. Johnny Raffoul, Catherine Lassabe, Christine Monnin-Devalland, Shan Ron Sun, Yannick Jeffredo, Etienne Faure, Olivier Depret, Frederic Ringenbach, Frederic Ansart, Micheline Chargeboeuf, Isabelle Bedgedjian, Alain Petitjean, and Adrien Khamlu for providing FFPE blocks. We thank Anne Duperrier for her technical assistance and Frances Sheppard (Clinical Investigation Centre, Besançon, France) for proofreading the article.

Footnotes

This work was supported by the Institut National du Cancer (INCa) and the University Hospital Centre of Besançon (Contrat d'Innovation 2009).

Supplemental material for this article can be found at http://jmd.amjpathol.org or at doi: 10.1016/j.jmoldx.2011.05.010.

Supplementary data

Supplemental Figure S1:

The pGEMT-G13D plasmid was used to detect the KRAS p.G13D mutation (nucleotide change c. 38G>A). SNaPshot sensitivity was ~1%. In contrast, the chromatograms on the right side showed that the mutant allele cannot be detected below the 5% level by direct sequencing. Representative sensitivity testing using mutant plasmids. DNA from mutant plasmids was diluted into wild-type plasmids as specified by the percentage values on the left. Mutant alleles are indicated by arrows. The section on the left represents the multiplexed SNaPshot assay. The middle section is a magnified image of the mutation detection. The section on the right represents tracings for reverse DNA strands obtained by sequencing analysis of the same samples. A no-DNA negative control is shown (bottom).

Supplemental Figure S2:

The pGEMT-G13R plasmid was used to detect the KRAS p.G13R mutation (nucleotide change c. 37G>T). SNaPshot sensitivity was ~5%. In the sequencing analysis, the mutation peak is not distinguishable from the background. Representative sensitivity testing using mutant plasmids. DNA from mutant plasmids was diluted into wild-type plasmids as specified by the percentage values on the left. Mutant alleles are indicated by arrows. The section on the left represents the multiplexed SNaPshot assay. The middle section is a magnified image of the mutation detection. The section on the right represents tracings for reverse DNA strands obtained by sequencing analysis of the same samples. A no-DNA negative control is shown (bottom).

Supplemental Figure S3:

The pGEMT-G12D plasmid was used to detect the KRAS p.G13D mutation (nucleotide change c. 35G>A). Sensitivity was ~1%. In contrast, the chromatograms on the right side showed that the mutant allele cannot be detected by direct sequencing when the mutant allele was present at 10%. Representative sensitivity testing using mutant plasmids. DNA from mutant plasmids was diluted into wild-type plasmids as specified by the percentage values on the left. Mutant alleles are indicated by arrows. The section on the left represents the multiplexed SNaPshot assay. The middle section is a magnified image of the mutation detection. The section on the right represents tracings for reverse DNA strands obtained by sequencing analysis of the same samples. A no-DNA negative control is shown (bottom).

Supplemental Figure S4:

The pGEMT-G12V plasmid was used to detect the KRAS p.G12V mutation (nucleotide change c. 35G>T). SNaPshot sensitivity was ~5%. In this example, when the mutant allele was present at 10%, it was distinguishable from the sequencing background because the site of mutation was known. When the frequency of the mutant was below 10%, the mutation was not detectable by sequencing (sections on the right). Representative sensitivity testing using mutant plasmids. DNA from mutant plasmids was diluted into wild-type plasmids as specified by the percentage values on the left. Mutant alleles are indicated by arrows. The section on the left represents the multiplexed SNaPshot assay. The middle section is a magnified image of the mutation detection. The section on the right represents tracings for reverse DNA strands obtained by sequencing analysis of the same samples. A no-DNA negative control is shown (bottom).

Supplemental Figure S5:

pGEMT-G12S plasmid was used to detect the KRAS p.G12S mutation (nucleotide change c. 34G>A). SNaPshot sensitivity was ~1%. At 1% of mutated allele, while the mutation codon 12 is definitely visible in the SNaPshot analysis, the mutation peak is hardly distinguishable from the background in the sequencing chromatogram. Representative sensitivity testing using mutant plasmids. DNA from mutant plasmids was diluted into wild-type plasmids as specified by the percentage values on the left. Mutant alleles are indicated by arrows. The section on the left represents the multiplexed SNaPshot assay. The middle section is a magnified image of the mutation detection. The section on the right represents tracings for reverse DNA strands obtained by sequencing analysis of the same samples. A no-DNA negative control is shown (bottom).

Supplemental Figure S6:

pGEMT-G12R plasmid was used to detect the KRAS p.G12R mutation (nucleotide change c. 34G>C). SNaPshot and sequencing sensitivity was ~1%. Representative sensitivity testing using mutant plasmids. DNA from mutant plasmids was diluted into wild-type plasmids as specified by the percentage values on the left. Mutant alleles are indicated by arrows. The section on the left represents the multiplexed SNaPshot assay. The middle section is a magnified image of the mutation detection. The section on the right represents tracings for reverse DNA strands obtained by sequencing analysis of the same samples. A no-DNA negative control is shown (bottom).

Supplemental Figure S7:

pGEMT-G12A plasmid was used to detect the KRAS p.G12A mutation (nucleotide change c. 35G>C). SNaPshot sensitivity was ~5% as that of sequencing analysis. Representative sensitivity testing using mutant plasmids. DNA from mutant plasmids was diluted into wild-type plasmids as specified by the percentage values on the left. Mutant alleles are indicated by arrows. The section on the left represents the multiplexed SNaPshot assay. The middle section is a magnified image of the mutation detection. The section on the right represents tracings for reverse DNA strands obtained by sequencing analysis of the same samples. A no-DNA negative control is shown (bottom).

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