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Keywords:

  • KRAS;
  • mutation detection;
  • colorectal cancer;
  • SNuPE;
  • HPLC

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Mutations in the KRAS gene are very important diagnostic and prognostic markers in cancer. Particularly, KRAS mutations at codons 12 and 13 have a high prognostic value for EGFR-directed antibody therapies. Several methods are available to detect the most common mutations, some of them are commercialized. The most frequently used techniques, allele-specific PCR or direct sequencing, are not standardized and often lack sensitivity to detect low amounts of mutated tumor cells in paraffin-embedded tissue-blocks leading to a high number of false-negatives. Here we present a reliable, fast, cost-effective and sensitive approach for KRAS mutation detection that has a high potential for standardized large scale screening. The method is based on multiplexed primer extension reactions coupled to HPLC separation. The highly sensitive assay gives easily interpretable and reproducible results at affordable costs. We describe the method and an application example for diagnosis in early colorectal cancer screening.

KRAS is a member of the family of small G proteins involved in the transmission of growth regulatory signals from the cell membrane to intracellular signalling networks. KRAS is activated by tyrosine kinase receptors such as EGFR or PDGFR.1, 2 Upon mutation, the KRAS protein can be activated but the switch from the active to its inactive form is prohibited resulting in continous mitogenic signaling and cell cycle progression.3 Numerous studies showed that mutations within KRAS codons 12 and 13 occur frequently (40%) in colorectal cancer patients and are of high predictive importance.4–6 The prognostic value of this marker, however, is still a matter of debate as outlined recently.7, 8 The introduction of epidermal growth factor receptor inhibitors (EGFR-I) for therapy in metastasized colorectal cancer recently revealed a strong predictive power of mutated KRAS. Only a subset of patients is sensitive to EGFR inhibitors9–11 and retrospective studies correlated the insensitive tumors with mutations in the KRAS gene.12 Including KRAS mutation screening into routine CRC diagnostics therefore helps to improve the selection of patients, which are promising candidates likely to benefit from EGFR-I treatment.

Several methods have been developed to detect KRAS mutations.13–20 Currently, the most widely used technique is direct sequencing, as it does not require complex and expensive chemicals and/or assay design. However, successful direct sequencing is highly dependent on template quality and quantity, which can be challenging upon working with formaldehyde-fixed paraffin-embedded tissue blocks and biopsies. Consequently, it turned out to be difficult to detect less than 20% of mutated tumor DNA within a tissue sample since signals do not rise beyond background noise. Thus, these samples might be classified as wild-type although they are not. To circumvent this, more sensitive and robust diagnostic methods are needed to detect mutated tumor DNA in an excess of wildtype cells in order to chose if the growing tumor is potentially sensitive to EGFR-I treatment or not.

Here we present a primer extension-based approach with subsequent IP/RP-HPLC separation that detects selectively all 12 clinically relevant variants (eight mutations and four wild-type bases) known for codons 12 and 13 of the KRAS gene. The assay does not require complex chemistry or labeling and is optimized to analyze 50 individual DNAs within 24 hr with a high detection accuracy. As the workflow can be almost entirely automatized, a minimum of human resources is required.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Tissue block preparation and histology

Preparation of DNA from FFPE samples was performed as described in detail recently.21, 22 Four 3-μm sections were cut from every tissue sample. The first slide was stained for H&E and a histopathologic diagnosis was rendered by a pathologist. Subsequently, the tumor area was marked and the percentage of tumor in relation to the whole block was documented. Distribution of different cell types in the marked area was evaluated and the percentage of tumor cells, stromal cells, necrosis, fat tissue and normal cells (either normal colon mucosa, normal liver or normal lung) was noted. Inflammation was graded as either none, weak, moderate or strong. In addition, every tumor was graded according to WHO criteria and the percentage of mucinous tumor areas was evaluated. When biopsies were referred, one additional tissue slide was prepared at the end to ensure that the respective unstained slides still contained tumor. DNA was prepared using the Tissue QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and DNA amount was estimated with the Nanodrop 1000 Spectrophotometer (Peqlab, Erlangen, Germany).

PCR and direct sequencing

Prior to direct sequencing PCRs were performed in 50 μl reaction volume in the presence of 3 mM Tris-HCl, pH 8.8, 0.7 mM (NH4)2SO4, 50 mM KCL, 2.5 mM MgCl2, 0.06 mM of each dNTP, 0.2 μM of each primer (F: 5′-aaggcctgctgaaaatgactg-3′, R: 5′-agaatggtcctgcaccagtaa-3′), 3 U HotFire DNA polymerase (Solis BioDyne, Tartu, Estonia) and 2.5 μl (∼100 ng) template. After the initial denaturation of 5 minutes at 95°C, 40 cycles of 94°C 1 min, 60°C 1 min and 72°C 1 min followed by a final extension of 72°C for 7 min were performed. Purified PCR products (8.5 ng) were used for sequencing with Big Dye Terminator Cycle Sequencing Mix v1.1 (Applied Biosystems) according to the manufacturer's instructions. Sequencing reactions were performed for both DNA strands with the PCR oligonucleotides (5 pM) as respective primers. Sequence analysis was done on a 3130 Genetic Analyzer, software Sequencing Analysis 5.2, (Applied Biosystems). The obtained files were aligned and examined for mutations in codons 12 and 13 of the KRAS gene by SeqScape 2.6 Software (Applied Biosystems).

Primer extension and HPLC separation

Fifty nanograms of genomic DNA were used as a template in a 30 μl reaction volume in the presence of 3 mM Tris-HCl, pH 8.8, 0.7 mM (NH4)2SO4, 50 mM KCL, 2.5 mM MgCl2, 0.06 mM of each dNTP, 3 U HotFire DNA polymerase (Solis BioDyne, Tartu, Estonia) and 1 μM primers. Five microliters of the PCR product were treated with 1 μl of ExoSAP (1:10 mixture of Exonuclease I and Shrimp Alkaline Phosphatase, USB) for 30 min at 37°C. To inactivate the ExoSAP enzymes, the reaction was incubated for 15 min at 80°C. Afterward, 14 μl primer extension mastermix (50 mM Tris-HCL, pH9.5, 2.5 mM MgCl2, 0.05 mM of all four ddNTPs, 3.6 μM of each SNuPE primer, 2.5 U Termipol DNA polymerase [Solis BioDyne, Tartu, Estonia)] was added. HPLC-purified primers have the following sequences: P12-1 5′-tagttggagct-3′, P13-2 5′-actcttgcctacg-3′ (put into one reaction), P12-2 5′-gttggagctg-3′, P13-1 5′-gcactcttgcctacgc-3′ (put into one reaction). Primer extension reactions were performed at 96°C for 2 min followed by 50 cycles 96°C/30 sec, 50°C/30 sec, 60°C/2 min. Separation of products was conducted at 50°C by continuously mixing buffer B (0.1 M TEAA, 25% acetonitril) to buffer A (0.1 M TEAA), either over 13 min: 21–29% (P12-1, P13-2) or 15 min:18–28% (P12-2, P13-1).

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Assay design

To date, 12 nucleotide variants are known for KRAS codons 12 and 13, eight of them being the most frequent ones.23 Interestingly, the mutations are limited to the first two bases of the codons leaving the third position unchanged (Fig. 1a). At codon 12, positions 1 and 2, named C12-1 and C12-2 throughout the text, the wildtype nucleotide G can be replaced by the nucleotides A, C or T. In codon 13, also two variants for the positions 1 (named C13-1) and 2 (named C13-2) are known: C13-1: G (WT) and T (tumor) and C13-2: G (WT) and A (tumor).

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Figure 1. Known mutations for KRAS codons 12 and 13 and assay design. (a) First 20 codons of wild-type KRAS cDNA are shown with all so far detected mutations in codons 12 and 13. (b) SNuPE primer positions and their possible extension products (extended base marked in red); primers detecting the base information in C13-1 and C13-2 anneal onto the top strand, primers detecting mutations in C12-1 and C12-2 anneal onto the bottom strand.

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Our assay is based on single nucleotide primer extensions performed with oligos that anneal with their 3′-end directly adjacent to the polymorphic base. In total, we designed four different primers to detect all mutations described. Primers P12-1 and P12-2 anneal to the bottom strand of the PCR product, and primers P13-1 and P13-2 anneal to the top strand (Fig. 1b). Primers are extended by ddNTPs according to the genotypic information at the polymorphic position (outlined with red in Fig.1b, for more details about primer design see Supporting Information). After the extension reactions, primers have different lengths and hydrophobicities (caused by nature of the incorporated base) making them suitable for separation on an IP/RP-HPLC (here: WAVE™ system, Transgenomic). Detection was rendered by UV light; additional labeling or signal enhancement was found to be unnecessary. The primers were designed such as being suitable to multiplex the reaction and, subsequently, the HPLC run. We optimized the assay in terms of time- and cost-effectiveness by performing the extensions of primers P12-1 and P13-1 in one reaction and one single HPLC run (Fig. 2a). Analogously, mutations at C12-2 and C13-2 were detected in one primer extension reaction and in the very same HPLC run (Fig. 2b). As depicted in Figure 2, the peak characteristics for the specific mutations clearly light up in case mutations are present as compared to the wild-type profile.

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Figure 2. Electropherograms showing the peak separation profile for the multiplexed primer extension/HPLC assay. (a) Simultaneous detection of all known genetic variants in C12-1/C13-1 and C12-2/C13-2 by mixing PCR products possessing WT and mutated alleles prior to SNuPE reaction; on top, the incorporated ddNTP during SNuPE and the respective base information is given. (b) Representative picture for WT KRAS showing all peaks representative for the wild-type genotype; P12-1, P12-2 = unextended primers C12-1 and C12-2, respectively, P13-1, P13-2 = unextended primers C13-1 and C13-2, respectively.

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Assay sensitivity

To test the sensitivity of our assay and to compare the efficiency for each potentially mutated position, we cloned KRAS amplicons obtained from colorectal cancer tissues harbouring both wild-type and mutated KRAS. After sequencing of individual clones, PCR products with mutated and WT genotype were measured, diluted and mixed from 1:1 to 1:100, which equals between 600 pg and 6 pg mutated KRAS in an up to 100-fold excess of WT background. By this method, the detection limit for each of the eight known mutations in codons 12 and 13 was defined. Table 1 shows that all mutations could be detected at a threshold of 1:20 (30 pg template including either mutation; this corresponds to 5% reliable detection limit vs. 20% reliable detection limit in direct sequencing). Interestingly, analysis of C12-1 was the most sensitive [1:100 (6 pg)], analysis of C13 the least sensitive reaction [1:20 (30 pg)]. To further demonstrate the advantage of the assay versus the common direct sequencing method, we analyzed DNA extracted from formaldehyde-fixed, paraffin-embedded tissues as this represents the common diagnostic application. In these samples, tumor cells are embedded into areas of normal tissue so that WT and mutated genotypes are detected simultaneously. Looking at all possible changes at codons 12 and 13, signals obtained after primer extension and HPLC separation were robust—irrespective of the tumor/WT cell ratios ranging from 10:90 to 70:30%. Comparing samples with tumor/WT cell ratios from 50:50 to 10:90% with direct sequencing results, we observed samples below 20% of tumor cells provide signals hardly above background noise in direct sequencing but show robust tumor-specific peaks down to 10% within the HPLC electropherogram (Fig. 3). This makes our approach a much more reliable tool in potentially detecting early stages of cancerogenesis or mutations in low amounts of tissue.

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Figure 3. Examples for KRAS double blind study results obtained by sequencing and Multiplex-SIRPH; percentage of tumor cells within a tissue block determined by immunostaining and the respective amino acid are shown on the left followed by the direct sequencing electropherogram; arrows within the HPLC diagrams highlight the position of the peaks significant for the respective mutation, the arrow at the G/C polymorphic position C12-1 (12-Arg) indicate ambiguity when interpreting direct sequencing results; P12-1, P12-2 = unextended primers for C12-1 and C12-2, respectively, P13-1, P13-2 = unextended primers for C13-1 and C13-2, respectively. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Table 1. Detection limits obtained after spiking mutated template into WT background prior to PCR
inline image

Assay performance

To test the performance of our assay on clinical samples we analysed DNAs of 50 CRC biopsies for KRAS mutations in a blinded study comparing direct sequencing to the HPLC assay. DNAs were prepared from FFPE tissue blocks. Histopathology of these tissue blocks was assessed by immunostaining revealing a tumor cell content ranging from 10 to 60%. On all samples, we performed direct sanger sequencing and primer extension in independent labs without cross-information. Hence the results were obtained in parallel from the same DNAs following separate PCR amplifcation. As shown in Table 2, in all 50 cases WT and mutated samples were unambiguously assigned by our HPLC and direct sequencing, i.e., our assay reaches at least the same specificity and sensitivity as optimal direct sanger sequencing.

Table 2. Summary of the KRAS double blind study on primary colorectal epithelial tissue
inline image

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Here we present a novel, HPLC-based approach for high throughput screening of KRAS mutations reaching at least the specificity obtained by direct sequencing. The assay is simple, cost-effective and fast. Using two multiplexed reactions the whole mutation spectrum at codons 12 and 13 of KRAS could be unambiguously assigned in two HPLC runs at a third of the costs of direct sequencing. As the procedure is semi-automatable the genotyping of 50 DNAs in a 96-well format took only 24 hr. The assay is sensitive and robust. Following successful PCR, none of the samples had to be repeated because of insufficient signal quality or quantity.

In predictive clinical diagnostics, such fast, robust and reliable methods are of great value as therapy decisions are made on the basis of an accurate KRAS mutation status.9–11 Currently, direct sequencing is the most frequently used method, but it often suffers from limited sensitivity and discriminatory power after peak calling (base line problems), particularly when the amount of tumor cells within a tissue is lower than 20%. Moreover, qualitatively, the assignment of mutations is much more unambiguous in HPLC profiles compared to direct sequencing profiles (see 12-Arg profile for illustration). Alternatively, appropriate kits using pyrosequencing or the ARMS/S method are available. In diagnostic labs, these kits are hardly used as they are very expensive. Technically, pyrosequencing reaches similar sensitivity compared to our method (5% tumor in WT background), however, accuracy is diminished as shown in recent studies on formalin-fixed, paraffin-embedded samples.6, 24 This uncertainty leaves a number of potential false negatives. ARMS/S reaches a higher accuracy but, in terms of sensitivity, it is not superior to our method as it was calculated to the range of 4–8%.25 With our assay, the complete mutation spectrum can be detected with high accuracy and sensitivity to a minimum of costs.

A reliable and sensitive qualitative detection of KRAS mutations is important for the choice of therapy. Our method can also be used to quantify the amount of mutated alleles. We routinely use the method for bisulfite-based relative quantification of DNA methylation (formerly published by EL-Maarri in 2004), which requires calibration curves generated for individual primer extension products.26

Compared to other recently upcoming sensitive methods like pyrosequencing,27 real-time PCR,28 ARMS/S25 or LCN-HRM,29 primer extension/HPLC does not require complicated establishment procedures, expensive lab automation or technical replicates. In fact, the most critical point is the primer design since dimerization or self-annealing during SNuPE reaction, especially in multiplexed primer extension reactions, may lead to false positive results. Including no template controls in the primer extension step helps to avoid any non-specific extension. Primer separation can potentially be established on every HPLC system when using the DNASep™ column and can be rapidly conducted without extensive training.

Multiplexed primer extension/HPLC assays can in principle be adopted to all known single base variants of cancer related genes e.g., such as BRAF or PIK3CA. This approach even allows combining assays at different genes in one reaction/HPLC run to limit costs and processing time. The assay will be a fast method to screen potential new candidates arising from the large international cancer genome sequencing projects for their clinical prevalance in larger cohorts.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Dr. Christiane Schewe for providing the DNA from cancer samples and critical reading of the manuscript.

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  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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