Lasse Sommer Kristensen

Iben L Daugaard

Mariann Christensen

Stephen Hamilton- Dutoit

Henrik Hager

Lise Lotte Hansen

Increased sensitivity of KRAS mutation detection by High-Resolution Melting analysis of COLD-PCR products Running head: KRAS mutation detection by HRM analysis of COLD-PCR products

Keywords: Cancer, KRAS mutations, COLD-PCR, High-Resolution Melting Cancer, KRAS mutations, COLD-PCR, High-Resolution Melting EGFR, epidermal growth factor receptor, HRM, high-resolution melting, COLD-PCR, coamplification at lower denaturation temperature-PCR, T m , melting temperature, FFPE, formalin fixed paraffin embedded, PB, peripheral blood, dNTP, deoxynucleoside triphosphate, T c , critical denaturation temperature

Considerable effort has been invested in the development of sophisticated technologies enabling detection of clinically significant low-level tumor specific KRAS mutations. Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that selectively amplifies mutation-containing templates based on the lower melting temperature of mutant homoduplexes versus wild type homoduplexes. We have developed a fast COLD-PCR and High-Resolution Melting (HRM) protocol to increase the sensitivity of KRAS mutation detection. The clinical applicability of COLD-PCR for KRAS mutation detection was assessed by analyzing 61 colorectal cancer specimens, for which KRAS mutation status has been evaluated by the FDA approved TheraScreen ® KRAS mutation kit. The sensitivity was increased by 5 to 100 fold for melting temperature decreasing mutations when using COLD-PCR as compared to standard PCR.

Mutations, undetectable by the TheraScreen ® kit in clinical samples, were detected by COLD-PCR followed by HRM and verified by sequencing. Finally, we have observed a previously undescribed low prevalence synonymous mutation (KRAS c.39C>T, codon 13) in colorectal cancer specimens and in the peripheral blood from an unaffected individual. In conclusion, COLD-PCR combined with HRM is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments.

Introduction

Activation of the epidermal growth factor receptor (EGFR; MIM# 131550) stimulates proliferation, angiogenesis, invasion, and survival of cancer cells. Since EGFR is frequently overexpressed in a range of solid tumors it represents an important target for cancer treatment. Monoclonal antibodies targeting EGFR such as panitumumab and cetuximab have proved to significantly increase survival rates of a subgroup of metastatic colorectal cancer patients. However, immunohistochemical staining for the EGFR protein has been found to be a poor biomarker for predicting response to EGFR inhibition [START_REF] Chung | Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry[END_REF]. In contrast, activating mutations in the KRAS gene (KRAS; MIM# 190070) have proven to be a biomarker for non-reponse to EGFR blockage, and both the U.S. Food and Drug Administration (FDA) and the European Medicines Agency require that KRAS mutation status is determined prior to anti-EGFR treatment. The biological explanation is that KRAS is part of a mitogenic signaling cascade normally triggered by binding of EGF or other ligands to EGFR, and that oncogenic KRAS is able to transmit the signal independent of EGFR.

Screening for KRAS mutations is not only clinically relevant in metastatic colorectal cancer. The detection of KRAS mutations may be used for monitoring residual disease after treatment in lymphoma patients [START_REF] Cikota | Detection of t(14;18), P53 and RAS gene mutations and quantification of residual disease in patients with B-cell non-Hodgkin's lymphoma[END_REF], and evidence is also present that KRAS mutation status is highly predictive of response to anti-EGFR treatment in lung cancer [START_REF] Massarelli | KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer[END_REF].

In general, tumor heterogeneity as well as contamination with normal cells in clinical samples often results in clinically relevant mutations being diluted in wild type sequences below the detection limit of conventional PCR based assays. Furthermore, highly sensitive methods are required for the detection and identification of somatic mutations in early tumorigenesis and the emergence of resistance mutations (Milbury, et al., 2009b). Traditional Sanger sequencing has been considered the gold standard for detecting and identifying genetic alterations. However, this approach suffers from a relatively low sensitivity as mutant alleles must be present in a proportion of at least 10-20% to be reproducibly detected [START_REF] Vogelstein | Digital PCR[END_REF].

A range of different PCR-based technologies have been developed with the goal of increasing the analytical sensitivity of mutation detection [START_REF] Do | Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies[END_REF][START_REF] Newton | Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS)[END_REF][START_REF] Ogino | Sensitive sequencing method for KRAS mutation detection by Pyrosequencing[END_REF][START_REF] Takeda | Detection of K-ras mutation in sputum by mutant-allele-specific amplification (MASA)[END_REF][START_REF] Ward | Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples[END_REF]. However, increased sensitivity usually comes with the cost of lower accuracy, increased complexity, and higher costs (Milbury, et al., 2009b).

When applying a particular technology for routine diagnostic testing it must achieve a sufficient sensitivity without compromising accuracy, while being convenient and cost-efficient.

High-resolution melting (HRM) analysis is a convenient and cost-effective way of analyzing genetic as well as epigenetic variations (Kristensen and Hansen, 2009;[START_REF] Kristensen | Sensitive Melting Analysis after Real Time-Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection[END_REF][START_REF] Krypuy | High resolution melting for mutation scanning of TP53 exons 5-8[END_REF][START_REF] Krypuy | High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer[END_REF][START_REF] Ma | Detection of KRAS mutations in colorectal cancer by highresolution melting analysis[END_REF][START_REF] Polakova | High-resolution melt curve analysis: initial screening for mutations in BCR-ABL kinase domain[END_REF][START_REF] Takano | Epidermal growth factor receptor mutation detection using high-resolution melting analysis predicts outcomes in patients with advanced non small cell lung cancer treated with gefitinib[END_REF][START_REF] Wittwer | High-resolution DNA melting analysis: advancements and limitations[END_REF][START_REF] Wittwer | High-resolution genotyping by amplicon melting analysis using LCGreen[END_REF][START_REF] Wojdacz | Rapid detection of methylation change at H19 in human imprinting disorders using methylation-sensitive high-resolution melting[END_REF]. However, the sensitivity of HRM analysis of standard PCR products has generally been found to be limited to mutant alleles present at 5%-10% in a background of wild type DNA [START_REF] Krypuy | High resolution melting for mutation scanning of TP53 exons 5-8[END_REF][START_REF] Krypuy | High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer[END_REF][START_REF] Polakova | High-resolution melt curve analysis: initial screening for mutations in BCR-ABL kinase domain[END_REF][START_REF] Takano | Epidermal growth factor receptor mutation detection using high-resolution melting analysis predicts outcomes in patients with advanced non small cell lung cancer treated with gefitinib[END_REF]. Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that significantly increases the sensitivity of mutation detecting assays without adding to the costs and complexity of the experiment [START_REF] Li | Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing[END_REF].

However, fast COLD-PCR does not enrich for mutations that retain or increase the melting temperature (T m ) of the amplicon [START_REF] Luthra | COLD-PCR finds hot application in mutation analysis[END_REF], unless the protocol is adjusted to facilitate the formation of mutation/wild type heteroduplexes. This approach is called full COLD-PCR and significantly adds to the time it takes to perform the experiments while it may only provide a moderately increased sensitivity when applied to KRAS mutation detection (Zuo, et al ). In general, mutation enrichment can be expected to be 5-8 fold for melting temperature retaining mutations and 3-5 fold for melting temperature increasing mutations when using full COLD-PCR as opposed to standard PCR. A thorough description of the differences between fast and full COLD-PCR can be found in [START_REF] Li | Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing[END_REF]. In the human genome approximately 70% of all encountered mutations, and 84% of the somatic mutations found in colon cancer, are melting temperature decreasing [START_REF] Li | Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing[END_REF].

We have applied fast COLD-PCR and HRM for the analysis of KRAS exon 2 codon 12 and 13 mutations. By using five different mutation-containing cell lines serially diluted into wild type DNA we have compared the sensitivity of the assay with HRM following standard PCR. Furthermore, we have analyzed a cohort comprising 61 colorectal cancer specimens that have been screened for KRAS mutations using the FDA approved TheraScreen ® KRAS mutation kit, and directly compared the results obtained using HRM after standard PCR and after COLD-PCR, respectively. Sanger sequencing was used to confirm the HRM results. In addition, we have applied a two round COLD-PCR protocol to verify subtle deviations in melting profiles.

Materials and Methods

Nucleotide and Codon Numbering

Nucleotide numbering reflects the cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference GenBank sequence NM_033360.2, according to the Journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon number 1.

Samples and DNA Extraction

Formalin-Fixed Paraffin Embedded (FFPE) blocks from surgical biopsies of 61 patients diagnosed with adenocarcinoma in colon were selected from the archives at the Institute of Pathology, Aarhus University Hospital. The specimens were between 2 and 10 years old. However, we have previously shown that PCR and HRM analysis can be successfully performed using FFPE specimens aged up to 30 years (Kristensen, et al., 2009). Specimens containing vital tumor cells were chosen by an experienced pathologist and no microdissections were performed prior to DNA extraction. For each sample, six tissue sections of 10 µm were transferred into a micro centrifuge tube and incubated in 1 ml of xylene in a thermomixer at 45°C and 800 rpm for 20 min. After centrifugation at 12,200 rpm for 5 min at room temperature, the supernatant was removed. This step was repeated once.

Subsequently, the tissue samples were washed in 1 ml of 99% ethanol using a thermomixer at 60°C and 800 rpm for 20 min. After centrifugation at 12,200 rpm for 5 min the supernatant was removed.

The washing procedure was repeated once. The samples were air dried in the thermomixer at 60°C for 20 min after adding one drop of acetone to each sample, and incubated with 150 µl Proteinase K (10 mg/ml) in the thermomixer at 55°C and 1,000 rpm over night. DNA was extracted using the QIAamp DNA FFPE Tissue Kit (QIAGEN) according to the manufacturer's instructions.

DNA from peripheral blood (PB), obtained from medical students of both sexes in their first year of Medical school, was extracted following a modified salt precipitation protocol as previously described [START_REF] Hansen | Molecular genetic analysis of easily accessible breast tumour DNA, purified from tissue left over from hormone receptor measurement[END_REF].

The Local Ethical Committee, Aarhus County, Denmark, approved this study. Five cell lines containing different KRAS mutations were used in this study; A549 (c.34G>A, codon 12), DLD-1 (c.38G>A, codon 13), LS174T (c.35G>A, codon 12), codon 12),and SW480 (c.35G>T,codon 12). The cell lines were cultured in RPMI 1640 culture media (Invitrogen, Breda, The Netherlands) and supplemented with 10% fetal bovine serum, 2mM Lglutamine, and 10,000 IU/ml penicillin, and 0.1 mg/ml streptomycin (both Leo Pharma, Copenhagen, Denmark) antibiotics. The cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO 2 . The cells were harvested by scraping and DNA extracted as described for PB with slight modifications; the centrifugation steps were carried out for 5 min at 13000 rpm instead of 15 min at 3000 rpm, and 1 volume of isopropanol was added instead of two volumes of ice cold ethanol.

Cell lines and dilution series

DNA from each of the KRAS-mutant cell lines was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and serially diluted into wild type DNA obtained from PB from medical students to the following fractions (100%, 50%, 10%, 5%, 2%, 1%, 0.5%, 0.2%, and 0.1%).

Primer design

The KRAS (GenBank accession number NM_033360.2 [http://www.ncbi.nlm.nih.gov/GenBank/]) primers were designed to immediately flank codon 12 and 13 of exon 2, to ensure that sequence variations outside codon 12 and 13 did not give rise to aberrant melting curves. The primers used were: 5'-gaatataaacttgtggtagttggagct-3' (forward primer) and 5'-atcgtcaaggcactcttgcctac-3' (reverse primer) giving an amplicon size of 56 bp. To confirm that the primers were unable to amplify from the KRAS1P pseudogene on chromosome 6 (GenBank accession number NC_000006.11 [http://www.ncbi.nlm.nih.gov/GenBank/]), SNP analysis of rs4516942:G>A were performed using primers specific for the pseudogene: 5'-agcggctcccagacctgct-3' (forward primer) and 5'ggaattctctattgttggatcatattgg-3' (reverse primer).

Standard PCR and HRM Conditions

PCR cycling and HRM analysis were performed on the Rotor-Gene 6000™ (Corbett Research, Sydney, Australia). SYTO ® 9 (Invitrogen) was used as the intercalating dye. The final reaction mixtures consisted of 50 ng of DNA, 1x PCR buffer, 2.5 mmol/L MgCl 2 , 200 nmol/L of each primer, 200 µmol/L of each dNTP, 5 µmol/L of SYTO ® 9, 0.5U of HotStarTaq (QIAGEN) (5U/µL) in a volume of 20 µL. The cycling protocol was initiated by one cycle at 95°C for 15 min, followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, 72°C for 10 s, and one cycle at 95°C for 1 min. HRM was performed from 65°C to 95°C, with a temperature increase at 0.1°C/s. Samples were analyzed in triplicates (cell line experiments) or in duplicates (colorectal cancer specimens).

COLD-PCR and HRM Conditions

The COLD-PCR cycling conditions were experimentally determined by adjusting the number of initial standard PCR cycles and the critical denaturation temperature (T c ) to ensure strong mutation enrichment while maintaining adequate amplification of all dilutions.

The COLD-PCR experiments were done using the same instrument and reaction conditions as the standard PCR and HRM protocol. The cycling protocol was initiated with one cycle at 95°C for 15 min, followed by 15 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s, 30 cycles of 80.5°C for 10 s, 60°C for 10 s, and 72°C for 10 s, and one cycle at 95°C for 1 min. HRM was performed

Data analysis

The Rotor-Gene 6000 Series Software version 1.7.87 supplied with the instrument was used to analyze the data. This was done by looking at the derivative of the raw data (Melting curve analysis), the normalized HRM and difference graphs (High resolution melting analysis). For the difference graphs a wild type sample was selected as reference.

Mutation analysis using the TheraScreen ® KRAS mutation Kit

All samples were analyzed using the TheraScreen ® KRAS mutation kit (DxS Diagnostic Innovations). This kit analyze the mutation status for the seven most commonly found KRAS exon 2 mutations (see Table 1) by a technology that combines ARMS ® (allele specific PCR) with Scorpions ® real-time PCR. The manufacturer has reported the sensitivity to be 1% mutant alleles in a wild type background if sufficient DNA input is used.

Two-round COLD-PCR

A two-round COLD-PCR protocol was developed to confirm mutations of very low prevalence.

The COLD-PCR products from the first round of COLD-PCR were diluted 100 fold and used as template in a second round of COLD-PCR using the same primers and conditions as for the first round of COLD-PCR except no standard PCR cycles were used. The comparisons of COLD-PCR

Sanger sequencing

Sequencing was performed on the ABI Genetic Analyzer 3130 XL (Applied Biosystems). To create a longer product of the KRAS codon 12 and 13 containing template, which could be sequenced successfully, a second amplification was performed with M13 tagged KRAS primers. These products were sequenced using the BigDye terminator kit v1.1 (Applied Biosystems) according to the manufactures' instructions with slight modifications. The single stranded PCR was performed using 1 µL of the BigDye terminator in a final volume of 10 µL. Sequencing of the amplicon generated by the primers specific for the KRAS1P pseudogene was done directly.

Results

The analytical sensitivity of COLD-PCR versus standard PCR

We have compared the analytical sensitivity of the COLD-PCR and HRM assay with standard PCR followed by HRM using serial dilutions of mutant DNA derived from cell lines into wild type DNA. Five different KRAS mutations were analyzed; c.34G>A, codon 12 (A549), c.35G>A, codon 12 (LS174T), c.35G>T, codon 12 (SW480), c.35G>C, codon 12 (RPMI-8226), c.38G>A, codon 13 (DLD-1). Three different T c 's were tested (80°C, 80.5°C, and 81°C) and we found 80.5°C to provide the overall strongest selectivity for mutant alleles. The sensitivity was increased by up to 100 fold for melting temperature decreasing mutations when using COLD-PCR as compared to 2. The A549 and SW480 cell lines are homozygous for the KRAS mutations whereas all the other cell lines are heterozygous for the respective KRAS mutations. To facilitate direct comparisons between the cell lines, sensitivities in Table 2 are given as percentages of mutant alleles (assuming all cells contained the mutation) and not as percentages of cell line DNA. Figure 1 shows HRM and sequencing results derived from standard PCR and COLD-PCR amplifications of the A549 cell line dilutions.

The reproducibility of the COLD-PCR and HRM assay

The reproducibility of the COLD-PCR and HRM assay was assessed by repeating the A549 cell line dilution experiment six times in triplicate for each dilution point. The fraction of the replicates for which the mutation could be detected for each dilution point for each of the experiments is shown in Supp. Table S1. At least two of three replicates containing 0.1% mutant alleles were detectable in four out of six experiments.

Detection of KRAS mutations in clinical samples

We have analyzed the KRAS mutation status in 61 colorectal cancer specimens using the FDA approved TheraScreen ® KRAS mutation kit, standard PCR followed by HRM, and COLD-PCR followed by HRM, respectively. Sequencing was used to confirm selected HRM results. In two cases (sample 49-50) mutations were only detectable by the TheraScreen ® kit and COLD-PCR, and in four cases (sample 51-54) mutations were only detectable by COLD-PCR. These mutations were all melting temperature decreasing mutations as expected. In two cases (sample 55-56) mutations were only detectable by the TheraScreen ® kit. These mutations were temperature retaining mutations, which could also be expected. Finally, in five cases (sample 57-61) discrepancies were observed between the TheraScreen ® kit and the other methods. In three of these cases (sample 57-59) mutations were observed by HRM and sequencing after standard PCR as well as after COLD-PCR. In the remaining two cases (sample 60-61) temperature decreasing mutations were detected by the TheraScreen ® kit but not by HRM or sequencing after standard PCR or COLD-PCR. An overview of the results can be found in Table 3, and Figure 2 shows selected HRM and sequencing results from cases for which the three different methods showed contradicting results. Furthermore, when using COLD-PCR a synonymous c.39C>T, codon 13 mutation, which to our knowledge has not been described before, was detected in 14 of the 61 colorectal cancer specimens.

Therefore, additional samples deviated from wild type when using COLD-PCR as opposed to standard PCR.

The COLD-PCR primers do not amplify the KRAS1P pseudogene

We have confirmed that the COLD-PCR primers were unable to amplify the KRAS1P pseudogene at chromosome 6. This was done by SNP analysis using HRM and sequencing with pseudogene specific primers to establish the genotype of rs4516942:G>A located only within the pseudogene at position 1 of codon 13 analogue to KRAS. The genotype frequencies for the SNP in the European group are; A/A: 0.450, A/G: 0.417, and G/G: 0.133. Fifteen of the colorectal cancer specimens were genotyped for rs4516942 and six were homozygous for the A allele (0.400), two were homozygous for the G allele (0.133), and seven were heterozygous (0.467). Since this mutation is melting temperature decreasing and not detected with the COLD-PCR primers we can conclude that the COLD-PCR primers do not amplify the KRAS1P pseudogene.

Two-round COLD-PCR

A two-round COLD-PCR protocol was developed to evaluate whether melting profiles with subtle deviations from wild type were due to intrinsic variation of the instrument or due to the presence of low-level mutations. Mutations that were initially discarded as intrinsic variation in the HRM analysis or as background in the sequencing analysis could be confirmed as real mutations by applying an extra round of COLD-PCR. This was possible as melting profiles of mutation containing samples are much easier to distinguish from wild type melting profiles after two rounds of COLD-PCR. DNA from A459 (c.34G>A, codon 12) diluted into wild type DNA was used for these experiments. Figure 3 shows representative HRM profiles and sequencing results derived after one and two rounds of COLD-PCR respectively. It can be observed that the G>A mutation was not detected in one of the 0.5% dilutions indicating that minor variation from reaction to reaction occur.

Finally, these experiments also showed that the synonymous c.39C>T, codon 13 mutation detected in 14 of the 61 colorectal cancer specimens also was present in PB from an unaffected individual.

Discussion

Changing a single parameter of the PCR, the denaturation temperature, can potentially increase the sensitivity of subsequent mutation detection assays by up to a hundred fold. This has been shown for traditional Sanger sequencing, pyrosequencing, MALDI-TOF, and RFLP based detection [START_REF] Li | Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing[END_REF], for HRM analysis (Milbury, et al., 2009a), and for TaqMan-based real-time PCR (Li, et al., 2009b). The denaturation temperature of COLD-PCR is referred to as the critical denaturation temperature (T c ) and the precise determination of this temperature has been considered to be "tricky" [START_REF] Luthra | COLD-PCR finds hot application in mutation analysis[END_REF]. We found that selecting a T c one degree below the melting temperature of the wild type PCR amplicon provided maximum sensitivity, and thus, in this case, confirmed what has been considered a rule of thumb for T c selection (Milbury, et al., 2009a). The number of initial standard PCR cycles is also important for optimal sensitivity. We found that approximately 10 cycles before the quantification cycle (C q ) ensured robust amplification while maintaining strong mutation enrichment.

When comparing COLD-PCR to standard PCR, a drastically increased sensitivity was observed for all melting temperature decreasing mutations. The sensitivity ranged from 0.1% to 1% mutant alleles in a wild type background when using an input of 50 ng DNA in the reactions. However, some run to run variation as well as replicate to replicate variation were observed when repeating the A549 cell line experiments (Supp. Table S1). Furthermore, the benefit from using COLD-PCR as opposed to standard PCR was highly dependent on the specific mutation (Table 2). In contrast, the sensitivity was not increased for the melting temperature retaining mutation (c35G>C, codon 12) present in the RPMI8226 cell line. This was expected as fast COLD-PCR protocols do not facilitate the formation of mutation/wild type heteroduplexes. The full COLD-PCR approach described earlier enriches all types of mutations based on the lower melting temperature of mutation/wild type heteroduplexes. This approach, however, requires substantial optimization, mutation enrichment may be modest, and a lengthy hybridization time is needed.

The superior sensitivity of COLD-PCR versus standard PCR was also reflected by our analysis of colorectal cancer specimens. In six of 61 cases (sample 49-54), mutations were detected by HRM and sequencing after COLD-PCR but not after standard PCR. All six mutations were melting temperature decreasing. The FDA approved TheraScreen ® KRAS mutation kit is more sensitive than standard PCR followed by HRM and sequencing. However, in four cases (sample 51-54) mutations detected by COLD-PCR followed by HRM and sequencing were missed by the TheraScreen ® kit. These mutations were also melting temperature decreasing, and not detected by standard PCR and HRM. In two cases (sample 55-56), mutations were only detected by the TheraScreen ® kit. These mutations were, as expected, melting temperature retaining. The sensitivity of the TheraScreen ® kit is reported by the company to be 1% depending on the total amount of DNA used (http://www.dxsdiagnostics.com). However, it has been reported that only a minority of diagnostic samples derived from FFPE tissues can be analyzed at this level of sensitivity [START_REF] Kotoula | Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics[END_REF].

Another interesting aspect of this study is that discrepancies that could not be explained by the relative sensitivities of the methods were observed between the TheraScreen ® kit and the other two methods in five cases (sample 57-61). In three of these cases (sample 57-59), scored as wild type by the TheraScreen ® kit, mutations were observed by HRM and sequencing after standard PCR as well as after COLD-PCR. Nevertheless, these mutations were detected by the TheraScreen ® kit, but at too high C q -values to meet the threshold for scoring them as positives (according to the manufactures instructions). In the remaining two cases (sample 60-61) temperature decreasing mutations were detected by the TheraScreen ® kit but not by HRM or sequencing after standard PCR or COLD-PCR.

Concordance between standard PCR and COLD-PCR was observed in 55 of 61 cases (90%), between standard PCR and the TheraScreen ® kit in 52 of 61 cases (85%), and between COLD-PCR and the TheraScreen ® kit in 50 of 61 cases (82%). Concordance between all three methods was observed in 48 of 61 cases (79%).

Similar degrees of disconcordance between the TheraScreen ® kit and a range of other methods including, Sanger sequencing, pyrosequencing, and HRM after standard PCR, and another FDA approved KRAS mutation kit, the TIB Molbiol KRAS mutation detection kit (LightMix Kit, TIB Molbiol) have been reported in a large multicenter blinded study [START_REF] Whitehall | A multicenter blinded study to evaluate KRAS mutation testing methodologies in the clinical setting[END_REF]. In this study, high false-positive rates were also observed for the TIB Molbiol kit when analyzing FFPE samples [START_REF] Whitehall | A multicenter blinded study to evaluate KRAS mutation testing methodologies in the clinical setting[END_REF].

It can be observed from Table 3 that most of the discrepancy between the methods appear to be caused by the c.38G>A, codon 13 mutation. However, there is no obvious explanation for this since the difference in sensitivity between standard PCR and COLD-PCR is less for this mutation compared the other mutations. In addition, this mutation was detected in one sample by the TheraScreen ® kit and not by HRM after standard PCR and COLD-PCR, but for two other samples the situation was the opposite.

All together, the results reflect the order by which each method meet their sensitivity limits and suggest an advantage of using COLD-PCR. That the frequency of detected KRAS mutations in clinical samples is influenced by the sensitivity of the method has previously been noted [START_REF] Kobunai | The frequency of KRAS mutation detection in human colon carcinoma is influenced by the sensitivity of assay methodology: a comparison between direct sequencing and real-time PCR[END_REF]. However, clinical decisions based upon very low prevalence mutations are controversial.

The costs and labor associated with each method are also important factors for routine clinical testing. HRM is a rapid, high-throughput, and inexpensive method that has performed well in other studies for both frozen and FFPE samples [START_REF] Whitehall | A multicenter blinded study to evaluate KRAS mutation testing methodologies in the clinical setting[END_REF]. However, Sanger sequencing is more expensive and a necessary quality control step for HRM based assays. Nevertheless, HRM followed by sequencing is still less expensive and less time consuming than the commercially available TheraScreen ® and TIB Molbiol kits [START_REF] Whitehall | A multicenter blinded study to evaluate KRAS mutation testing methodologies in the clinical setting[END_REF]. The switching from standard PCR to COLD-PCR does not add to the costs of the assay.

When using COLD-PCR we have detected a synonymous c.39C>T, codon 13 mutation in 14 of 61 colorectal cancer specimens. Therefore, more samples deviated from wild type when using COLD- PCR as opposed to standard PCR, and thus, the use of COLD-PCR would increase the sequencing load in clinical settings. This synonymous mutation is probably not clinically relevant, and may be a result of 5-methyl cytosine deamination since it is part of a CpG site. Such mutations are the most commonly found mutations in the human genome. We have also detected the synonymous c.39C>T, codon 13 mutation in PB from an unaffected individual, which suggests that it is not tumor specific. However, in most of the samples carrying this mutation, a small peak was observed in the electropherogram after sequencing, suggesting that the mutation often is present at very low level.

We developed a two-round COLD-PCR protocol to confirm that the synonymous C>T mutation, and other cases where mutations were present at low levels, were not merely background. First of all, we have observed that very subtle deviations of melting profiles are often reflected by very small peaks in the electropherograms, which in many cases would have been attributed to background. By applying an extra round of COLD-PCR it was confirmed that these subtle deviations actually reflected low-level mutations. After the second round of COLD-PCR melting profiles of mutation containing samples were much easier distinguished from wild type melting profiles, and the peaks in the electropherograms were clearly distinguishable from background. Two-round COLD-PCR has previously been described by Li et al (Li, et al., 2009a). However, they have used a second set of primers for the second round of COLD-PCR. By using the same primers as for the first round, we did not need to optimize an extra assay, and no initial standard PCR cycles were necessary.

The use of fast COLD-PCR and HRM for the detection of KRAS mutations in clinical samples has been evaluated in a parallel publication by Mancini and colleagues [START_REF] Mancini | The Use of COLD-PCR and High-Resolution Melting Analysis Improves the Limit of Detection of KRAS and BRAF Mutations in Colorectal Cancer[END_REF]. However, a different primer design was used and the sensitivity of their assay was only evaluated using a single cell line (CCRF-CEM) containing the c.35G>A mutation and reported to be 0.8% mutant alleles. Furthermore, the clinical samples were analyzed by PCR and COLD-PCR followed by HRM and sequencing, and no comparisons with the TheraScreen ® kit were provided. Finally, a two round COLD-PCR protocol to confirm very low prevalence mutations was not established, which in combination with a lower sensitivity may explain why the c.39C>T, codon 13 mutation was not detected by [START_REF] Mancini | The Use of COLD-PCR and High-Resolution Melting Analysis Improves the Limit of Detection of KRAS and BRAF Mutations in Colorectal Cancer[END_REF].

In conclusion, COLD-PCR combined with HRM is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments. However, clinically irrelevant mutations present at very low levels may be detected, which emphasizes the need for subsequent confirmation by sequencing. 3. A. HRM and sequencing results after COLD-PCR for a specimen for which the mutation was only detectable by the TheraScreen ® assay and COLD-PCR. B. HRM and sequencing results after COLD-PCR for a specimen for which the mutation was only detectable by COLD-PCR. In this case, the synonymous c.39C>T, codon 13 mutation was also detected. C. HRM and sequencing results after standard PCR for a specimen for which the mutation was only detectable by the TheraScreen ® assay. D. HRM and sequencing results after COLD-PCR for a specimen for which disconcordance between the TheraScreen ® kit and the other two methods could not be explained by the relative sensitivities of the methods. The percentages refer to mutant alleles in a wild type background using 50 ng DNA input in the reactions. Three different T c 's were tested when using COLD-PCR.

Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference GenBank sequence (NM_033360.2), according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1. Human Mutation

  95°C, with a temperature increase at 0.1°C/s. All samples were analyzed in triplicates (cell line experiments) or in duplicates (colorectal cancer specimens).

  the TheraScreen KRAS mutation kit were performed without the use of two-round COLD-PCR.

  whereas no increase in sensitivity was observed for the c.35G>C, codon 12 melting temperature retaining mutation. The results are listed in Table

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Selected HRM and sequencing results from Table 3. A. HRM and sequencing results

Figure 3 .

 3 Figure 3. Two-round COLD-PCR improves the separation between wild type and mutation

Figure 1 .Figure 2 .Figure 3 .Figure 1 .Figure 2 .Figure 3 .

 123123 Figure 1. Selected HRM and sequencing results from Table 2. The sensitivity was increased by approximately 100 fold for the G>A mutation present in the A459 cell line when using COLD-PCR instead of standard PCR. Selected replicates are shown. A. HRM and sequencing results after standard PCR. B. HRM and sequencing results after COLD-PCR. 254x190mm (600 x 600 DPI)

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Table 1 . KRAS exon 2 mutations detected by the TheraScreen ® KRAS mutation kit. KRAS hotspot mutations

 1

	Mutation	Amino acid change
	c.35G>C	Gly12Ala
	c.35G>A	Gly12Asp
	c.34G>C	Gly12Arg
	c.34G>T	Gly12Cys
	c.34G>A	Gly12Ser
	c.35G>T	Gly12Val
	c.38G>A	Gly13Asp
	Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG
	translation initiation codon in the reference GenBank sequence (NM_033360.2), according to
	journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.

Table 2 . Sensitivity of standard PCR versus COLD-PCR.

 2

Table 3 . KRAS mutation analysis of 61 colorectal cancer specimens.

 3 Consensus groups: A, complete agreement between all three assays. B, mutation only detectable by the TheraScreen ® assay and COLD-PCR. C, mutation only detectable by COLD-PCR. D, mutation only detectable by the TheraScreen ® assay. E, Disconcordance between the TheraScreen ® kit and the other two methods not explained by the relative sensitivities of the methods. The asterisk indicates that a synonymous c.39C>T, codon 13 mutation was detected.

	Cell line

John Wiley & Sons, Inc.Human Mutation

Acknowledgements

Tina Kjeldsen and Tina Hindkjaer are acknowledged for excellent technical assistance. Professor Torben Falck Ørntoft, Professor Peter Andreasen, and Dr. Bo Sandahl Sørensen kindly donated the cell lines used in this study. This study was funded by the Danish Cancer Society, the Lundbeck Foundation, the Foundation of A. P. Møller and Chastine McKinney Møller, and Max and Inge Wørzners Memorial Foundation.