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Clinical Chemistry 48: 2164-2170, 2002;
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(Clinical Chemistry. 2002;48:2164-2170.)
© 2002 American Association for Clinical Chemistry, Inc.

Pyrosequencing as an Alternative to Single-Strand Conformation Polymorphism Analysis for Detection of N-ras Mutations in Human Melanoma Metastases

Åsa Sivertsson1, Anton Platz2, Johan Hansson2 and Joakim Lundeberg1a

1 AlbaNova University Center, Department of Molecular Biotechnology, Royal Institute of Technology (KTH), S-106 91 Stockholm, Sweden.

2 Cancer Center Karolinska, Department of Oncology-Pathology, Radiumhemmet, Karolinska Hospital and Institute, S-171 76 Stockholm, Sweden.

aAuthor for correspondence. Fax 46-8-5537-8481; e-mail joakim.lundeberg{at}biotech.kth.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Mutations in codons 12, 13, and 61 of the N-ras gene are common alterations in cutaneous malignant melanoma. We evaluated pyrosequencing, a simple and rapid method used mainly for single-nucleotide polymorphism analysis, as a possible alternative to single-strand conformation polymorphism (SSCP) analysis and sequencing of N-ras.

Methods: We evaluated the sensitivity and accuracy of the pyrosequencing method for identification of mutations in N-ras codons 12, 13, and 61. Nucleotide dispensation orders were created to produce distinct pyrogram peak profiles for the most frequent mutations in codon 61 and codons 12 and 13, respectively.

Results: The detection limits for the two most common codon 61 mutations found in malignant melanoma, which code for Arg and Lys, were 30% and 15%, respectively. To evaluate the pyrosequencing method on clinical samples, we performed a parallel analysis of 82 melanoma metastases using SSCP analysis and pyrosequencing. All mutations detected by SSCP analysis and confirmed by sequencing were also correctly identified by pyrosequencing. Codon 61 mutations were identified in 26 of the 82 samples (32%), whereas no mutations were found in codons 12 and 13. Four types of codon 61 mutations, Arg (17%), Lys (10%), Leu (4%), and His (1%), were identified.

Conclusion: Pyrosequencing is an attractive alternative to SSCP analysis for N-ras mutation detection in malignant melanoma tumor samples because it displays the same sensitivity and accuracy as SSCP analysis and is simple and rapid.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Screening and identification of gene mutations in tumor material is commonly performed by PCR amplification followed by direct DNA sequencing or conformation-based separation such as single-strand conformation polymorphism (SSCP)1 analysis or denaturing gradient gel electrophoresis (1)(2)(3)(4)(5)(6). Direct sequencing, regarded as the gold standard, is an accurate and sensitive method that in its automated form is a very high-throughput technology. However, the cost per sample is rather high, and expensive equipment is required. The simplicity and low cost of alternative methods, such as SSCP analysis and denaturing gradient gel electrophoresis, have made them widely used for prescreening, but their sensitivity and specificity have been disputed. The need for a confirming nucleotide sequence analysis adds additional costs and time to the complete analysis.

Pyrosequencing is a real-time, nonelectrophoretic sequencing method that is rapidly gaining popularity for use in a large variety of applications (7)(8)(9)(10)(11)(12)(13). The method relies on the sequential addition and incorporation of nucleotides in a primer-directed polymerase extension reaction. The pyrophosphate released on incorporation of a base-paired nucleotide is coupled to the enzymes ATP sulfurylase and luciferase to generate detectable light. The light is proportional to the number of nucleotides incorporated, allowing for the sequence to be determined in real time. The resulting peak pattern will differ between homo- and heterozygous wild-type (wt) and mutant samples so that type and exact localization of the mutations can be determined. In a rapid, simple, and cost-effective manner, short stretches of sequence from a large number of samples are analyzed in parallel. The above-mentioned features in addition to the ability to distinguish between the different alleles make this method especially suitable for applications such as single-nucleotide polymorphism (SNP) analysis and hot spot mutation detection.

Activating mutations in the ras proto-oncogenes are among the most commonly found alterations in cutaneous melanoma, the most fatal of skin cancers. The ras family contains three functional proto-oncogenes N-ras, H-ras, and K-ras, which encode membrane-bound proteins belonging to the family of GTP-binding G-proteins. In the active GTP-bound state, these proteins stimulate extracellular growth and differentiation by transduction of signals through several effector pathways (14). The signals are switched off when the intrinsic GTPase activity turns the protein into its inactive GDP-bound state. Mutations that lock the protein in the active state and thus produce continuous activation of downstream targets commonly occur in tumorigenesis. Activating mutations in the ras family of proteins preferentially occur in codons 12, 13, 61, and 63 (15) and exert their effect by reducing the intrinsic GTPase activity of the protein in addition to making it insensitive to GTPase-activating proteins (16)(17)(18). Different tumor types are typically associated with activating mutations in a particular ras gene. In the case of melanoma, the vast majority of mutations are found in N-ras, and the incidence reported is between 5% and 36% [Refs. (19)(20)(21)(22)(23) and Omholt et al., unpublished data]. Codon 61 of exon 2 is the most commonly mutated because of preferential formation of pyrimidine dimers at this site after exposure to ultraviolet light (24)(25).

In this study, pyrosequencing was compared with SSCP analysis/direct sequencing, in terms of accuracy, sensitivity, and detection limit, for screening of mutations in N-ras codons 12, 13, and 61 in frozen samples from melanoma metastases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
samples and dna extraction
Fresh-frozen surgical biopsies of 82 lymph node and soft tissue metastases from melanoma patients monitored at the Department of Oncology, Karolinska Hospital, were included in this comparative study of the two mutation detection methods. Another 17 tumor samples were used to evaluate different DNA extraction methods. Frozen material was cut in 20-µm sections, and the tumor tissue was manually dissected after eosin–hematoxylin staining to enrich for tumor cells. The DNA was then extracted by either proteinase K treatment (1 µg/µL proteinase K in 1x Tris-EDTA at 60 °C for 24 h, followed by heating at 95 °C for 15 min) or by use of the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instruction.

To determine the detection limit for pyrosequencing, we used two cell lines containing two different N-ras codon 61 mutations as well as normal blood containing wt N-ras. The human melanoma cell line 224 is hemizygous (and thus contains 100% mutated DNA) for a mutation in codon 61, corresponding to a Gln (CAA) to Arg (CGA) amino acid change, whereas the human fibrosarcoma cell line HT-1080 is heterozygous (and contains 50% mutated and 50% wt DNA) for a mutation in the same position corresponding to a substitution of Gln (CAA) for Lys (AAA).

pcr amplification
For use in pyrosequencing, two fragments, 103- and 136-bp long (from exons 1 and 2, respectively), were PCR amplified in a multiplex or in single fashion. The multiplex PCR was followed by an inner nested PCR for exon 1 and a seminested PCR for exon 2. PCR reactions contained 1x PCR buffer II (Perkin-Elmer), 0.2 mM each deoxynucleotide triphosphate, 2 mM MgCl2, 0.3 µM each primer, and 0.25 U of AmpliTaq DNA polymerase per 10-µL reaction. To improve amplification of samples containing substantial amounts of melanin, we added 2 µg of bovine serum albumin per 10-µL PCR reaction. The primers used in the multiplex outer PCR were as follows: for exon 1, 5'-gatgtggctcgccaattaccctg-3' and 5'-accactgggcctcacctctatgg-3'; for exon 2, 5'-gattcttacagaaaacaagtggttatagat-3' and 5'-caaatgacttgctattattgatggca-3'. For the inner and single PCR reactions, the following primers were used: for exon 1, 5'-ggtgtgaaatgactgagtacaaactgg-3' and 5'-biotin-catattcatctacaaagtggttctgga-3'; for exon 2, 5'-gattcttacagaaaacaagtggttatagat-3' and 5'-biotin-gcaaatacacagaggaagccttcg-3'. In the outer multiplex and single amplifications, samples were denatured at 94 °C for 5 min; amplified for 30 cycles consisting of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min; and finally extended at 72 °C for 10 min. The inner PCR after the multiplex was performed as above except that the number of cycles was decreased to 20. All PCR reactions contained several samples without DNA as negative controls, as well as a N-ras wt sample.

For use in SSCP analysis, an 89-bp fragment of exon 1 and a 118-bp fragment of exon 2 were amplified in a seminested and nested fashion, respectively. The primers used in the outer PCR were as follows: for exon 1, 5'-atgactgagtacaaactggt-3' and 5'-ctctatggtgggatcatatt-3'; for exon 2, 5'-gattcttacagaaaacaagtg-3' and 5'-atgacttgctattattgatgg-3'. For the inner amplification, the following primers were used: for exon 1, 5'-atgactgagtacaaactggt-3' and 5'-atctacaaagtggttctgga-3'; for exon 2, 5'-caagtggttatagatggtga-3' and 5'-atacacagaggaagccttcg-3'. The reaction mixture contained 1x PCR buffer, 2.5 mM MgCl2, 0.2 mM each deoxynucleotide triphosphate, 5 pmol of primer, and 0.25 U of AmpliTaq polymerase (Applied Biosystems). The outer amplification was performed as above except that the annealing temperature was 56 °C. The conditions for the inner PCR was exactly as above, except that 3 µCi of [{alpha}-32P]dCTP was included for labeling the product.

mutation analysis
SSCP analysis and cycle sequencing.
PCR products were denatured in denaturing buffer (950 mL/L formamide, 10 mmol/L NaOH, 0.5 g/L bromphenol blue, 0.5 g/L xylene cyanol) at 92 °C for 10 min before electrophoresis on 7.5% nondenaturing acrylamide gels in the presence of 100 mL/L glycerol at 18 °C for exon 2 and without glycerol at 4 °C for exon 1.

Shifted bands were excised, reamplified, and purified using the QIAquick Gel Extraction Kit (Qiagen). Sequence analysis was then performed on an ABI 377 automated DNA sequencer (Applied Biosystems) with the BigDye Terminator Cycle sequencing reagent set (Applied Biosystems) and the inner primers as sequencing primers. Mutations were confirmed by two independent PCR/SSCP and sequence analyses.

Pyrosequencing.
The biotinylated products of the inner or single PCRs were immobilized on streptavidin-coated paramagnetic beads (Magnetic Biosolutions), and the strands were separated using 0.10 mol/L NaOH. The supernatant was then discarded, and the pyrosequencing primer (5 pmol) was added and annealed to the captured strand. The biotinylated DNA with annealed sequence primer was released from the streptavidin surface according to the manufacturer’s instructions (Magnetic Biosolutions). The sequences of the pyrosequencing primers were as follows: for codons 12 and 13, 5'-caaactggtggtggttggag-3'; for codon 61, 5'-gacatactggatacagctgg-3'. The above reactions were performed in 96-well plates in an automated fashion using the MBS robot (Magnetic Biosolutions). The primed single-stranded DNA templates were then transferred to the microtiter plate-based PSQTM Pyrosequencer (Pyrosequencing), where real-time sequencing of the sequence surrounding codons 12 and 13 of exon 1 and codon 61 of exon 2, respectively, was performed with the PSQ 96 SNP Reagent Kit (Pyrosequencing). The optimal nucleotide dispensation order was determined at the desk by looking at the theoretical outcome of several dispensation orders. The theoretically preferred order was then used on the pyrosequencer with no further optimization required. A software already exists that suggests dispensation orders for the typing of SNPs, and a similar software for detection of selected types of mutations affecting an entire codon will probably soon be available. Mutations were confirmed by pyrosequencing of an independent PCR reaction.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
evaluation of template preparation and the pcr for pyrosequencing
Before analyzing clinical samples, we optimized and evaluated different steps of the pyrosequencing method. We first evaluated two different methods for obtaining template DNA. From 17 different tumor samples, we prepared both pure DNA extract, obtained with the QIAamp DNA Mini Kit, and crude extract by proteinase K treatment. Amplification and pyrosequencing of the N-ras exon 1 and 2 fragments revealed no difference in either amplification efficiency or pyrosequencing results when the different extracts were used as template (Fig. 1 ). In addition, we developed a multiplex nested PCR approach for amplification of exons 1 and 2 of N-ras, which had the advantage that each outer PCR yielded template for several inner amplifications and, thus, for several pyrosequencing reactions.



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  		Figure 1. Pyrograms of exon 2 obtained with cyclic dispensation using crude DNA extract from proteinase K lysis and pure DNA extract from QIAprep extraction, respectively, as template.

evaluation and optimization of the pyrosequencing method
A cyclic nucleotide dispensation approach with iterative addition of the nucleotides as well as a programmed approach was evaluated. The programmed approach, in which the nucleotides are added according to the sequence context, showed an increased potential for achieving high resolution of different mutations and was therefore selected for use in this study (data not shown). The nucleotide dispensation order was optimized to give pyrogram peak profiles where wt (Gly), Arg, Ser, Asp, and Val at positions 12 and 13 of exon 1, and wt (Gln), Arg, Lys, Leu, and His at position 61 of exon 2 could be clearly distinguished (Fig. 2 ). The detection limit of pyrosequencing for this application was then determined using two cell lines containing different N-ras codon 61 mutations. Cell line 224 has a Gln-to-Arg change in codon 61 corresponding to 100% mutated DNA, because of loss of heterozygosity, whereas the cell line HT-1080 displays a heterozygous mutation corresponding to a Gln-to-Lys substitution in the same position. DNA from the cell lines was separately diluted with normal blood DNA (containing wt N-ras) to obtain mutant/wt ratios of 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, and 0% for the two different mutations. In addition, mutant/wt ratios of 100%, 75%, and 60% were obtained for the Arg variant from DNA from the 224 cell line. The samples were amplified, and the subsequent pyrosequencing, using an optimized nucleotide dispensation order, revealed detection limits of 30% for the codon 61 mutation that produced an Arg substitution (data not shown) and 15% for the mutation that produced a codon 61 Lys alteration (Fig. 3 ). The peak-height ratio between a calibration peak (constant signal) and a peak differing between wt and mutant samples was calculated for all samples. For each sample, the obtained value was then divided by the value obtained for the wt sample. The results demonstrated a linear relationship between peak signal and percentage of mutant to wt DNA.



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  		Figure 2. Nucleotide dispensation orders used to produce specific pyrogram peak profiles for the most common mutations at N-ras codons 12 and 13 in exon 1 (A) and codon 61 in exon 2 (B).

The sequences covered by pyrosequencing are displayed above the tables with the variant codons underlined together with a pyrogram of the wt sequence.



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  		Figure 3. Pyrogram peak profiles for different dilutions of the sample containing the mutation producing a Lys in codon 61.

The boxes indicate the positions distinguishing the Lys mutation from the other mutations as well as the wt; the arrows indicate all positions distinguishing the Lys from a wt sample. Shown to the right are the frequencies of the mutated and the wt allele, respectively, in the samples.

mutation analysis of melanoma metastases using pyrosequencing and sscp analysis/cycle sequencing
Mutation analysis of exons 1 and 2 of the N-ras gene was performed on DNA extracts from 82 melanoma metastases, using pyrosequencing and SSCP analysis/cycle sequencing in a parallel blind fashion. All samples were successfully amplified under the pyrosequencing PCR conditions, whereas 11 samples failed to amplify under the PCR conditions developed for SSCP analysis. Six samples contained very large amounts of melanin and were successfully amplified for pyrosequencing analysis only after electrophoretic separation of DNA and melanin according to the method of Price and Linge (26). At least three pyrosequencing analyses and two SSCP analyses using the products of different PCR reactions were performed on each sample to investigate the reproducibility. All results were consistent. The SSCP analysis showed bandshifts indicating mutations in 22 of the 71 amplified samples (31%) from exon 2. From comparisons of the relative intensities of the shifted bands to the wt bands, the concentrations of the mutated alleles were estimated as 10–40%. Sequencing confirmed and identified the mutations as corresponding to the following amino acid changes: 12 Arg (17%), 7 Lys (10%), 2 Leu (3%), and 1 His (1.5%), all in codon 61 (Table 1 ). All of the mutations found in the 71 samples subjected to SSCP analysis and sequencing were also clearly detected and correctly identified by pyrosequencing. Another four mutations, of which two corresponded to Arg substitutions, one to a Lys, and one to a Leu alteration, were detected among the 11 remaining samples analyzed by pyrosequencing alone. Thus, 26 of the 82 samples analyzed by pyrosequencing (32%) contained a codon 61 mutation, with the majority producing Arg (17%) followed by Lys (10%), Leu (4%), and His (1%) substitutions (Table 1 ). No mutations in codons 12 and 13 were detected by either of the methods.


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  		Table 1. Mutations detected and identified in N-ras codon 61 by pyrosequencing and SSCP analysis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we evaluated pyrosequencing as an alternative method to SSCP analysis for mutation screening of N-ras codons 12, 13, and 61 in malignant melanoma metastases. SSCP analysis is a widely used method for mutation detection that has the advantages of low cost and high sensitivity, but it also is heavily dependent on the analytical conditions and therefore presents difficulties in terms of reproducibility. The need to confirm the mutations by excising, reamplifying, and sequencing the bandshifts makes this method rather time-consuming and subjects the samples to three rounds of PCR amplification before sequencing, which is not optimal. Pyrosequencing, in contrast, is a real-time sequencing method that uses enzymatic reactions to monitor the inorganic phosphate released on nucleotide incorporation. Thus, the method avoids labeled nucleotides and is therefore more straightforward than SSCP analysis. In addition, pyrosequencing is time- and cost-competitive, having the ability to analyze 96 samples in <20 min and with a cost of less than US $1.00/sample. It has been shown to be an optimal method for SNP analysis where the allele frequency is 50% and the nucleotide variants are known (8)(13)(27). In tumor material, however, the mutated allele can be present at a frequency <50% because of infiltration by nontumor cells and tumor heterogeneity. Furthermore, hot spot mutations at a particular codon may affect all the different nucleotides and may represent a variety of different amino acid changes at each position. These features can be addressed by pyrosequencing technology. Here we demonstrate optimization of two nucleotide dispensation profiles, one that distinguishes among the five most common mutations at codon 61 and one that distinguishes among the seven most common mutations at codons 12 and 13, for analysis of N-ras hot spot mutations in 82 melanoma metastases. Using these profiles, we were able to detect and correctly identify all mutations found by SSCP/nucleotide sequence analysis of the same material. A total of 26 mutations were detected among the 82 metastases samples. This mutation frequency, 32%, correlates well with previous studies [Refs. (19)(28) and Omholt et al., unpublished data].

In SSCP analysis, detection of a mutation present in as little as 10% of the sample is possible by sequencing of the excised and reamplified bandshift (Omholt et al., unpublished data). The detection limit of automated direct sequencing is, from previous experience, between 10% and 40%, depending on the system used. The former estimate was obtained by use of slab gel-based electrophoresis and dye-labeled primer, whereas the latter represents results with capillary electrophoresis instrumentation and dye-terminator chemistry (data not shown) (29)(30). The pyrosequencing mutation detection limit is dependent on how well a dispensation profile can be created, which in turn depends on the nucleotide change resulting from the mutation as well as the adjacent nucleotide sequence. In this study, we determined that the detection limits for the two most common codon 61 mutations were 15% and 30%, respectively, which for most applications are satisfactory. Apparently the sensitivity was sufficient to detect two other codon 61 mutation types (Leu and His) present in our material, which from the SSCP gel band intensities were estimated to be present at concentrations of 20–30%.

A limitation with pyrosequencing when applied to mutation screening is that only a few positions can be analyzed simultaneously, whereas SSCP analysis essentially scans the entire amplified DNA fragment. In the present study, this was not an issue because most N-ras mutations in sporadic malignant melanoma affect codons 12, 13, and 61 (23)(31). For such mutation hot spots, pyrosequencing provides the possibility to design a nucleotide dispensation order that increases the sensitivity of detection in combination with identification of all the relevant mutation variants. In this study, we designed the dispensation orders manually with the goal to achieve as high a sensitivity as possible for detection of the included mutation variants. The sensitivity relates to the ease of distinguishing the pyrogram peak pattern resulting from a certain type of mutation from those of the other types at the lowest possible frequency of the mutated allele. For example, the difference in peak height between 2 and 2.5 peak equivalents is more difficult to detect than that between 0 and 0.5. In addition, if differences at multiple positions in the pyrogram can be achieved (Fig. 2Up ), the sensitivity increases further.

In conclusion, we find pyrosequencing to be a rapid, simple one-step method for performing sensitive and accurate detection/identification of N-ras hot spot mutations; it thus is a very attractive alternative to SSCP analysis for this application. Our recent finding that codon 61 N-ras mutations, when present in a primary melanoma, persist during tumor progression and can be identified in essentially all metastases in these patients makes this genetic alteration an interesting target for future therapeutic trials (Omholt et al., unpublished data). Development of such novel therapies will require large-scale screening of melanoma patients for N-ras mutations, and this may be an important future application of the pyrosequencing technology described here.


   Acknowledgments
 
We are grateful to Doris Kröckel, Eva Grafström, and Marianne Frostvik for excellent technical assistance. This work was supported by grants from the Swedish Cancer Society, the Swedish Scientific Council, the Cancer Society of Stockholm, the King Gustav V Jubilee Fund, the Karolinska Institute Research Funds, and the Swedish Radiation Protection Institute.


   Footnotes
 
1 Nonstandard abbreviations: SSCP, single-strand conformation polymorphism; wt, wild type; and SNP, single-nucleotide polymorphism.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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S. Ogino, T. Kawasaki, M. Brahmandam, L. Yan, M. Cantor, C. Namgyal, M. Mino-Kenudson, G. Y. Lauwers, M. Loda, and C. S. Fuchs
Sensitive Sequencing Method for KRAS Mutation Detection by Pyrosequencing
J. Mol. Diagn., August 1, 2005; 7(3): 413 - 421.
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K. E. Kruckeberg and S. N. Thibodeau
Pyrosequencing Technology as a Method for the Diagnosis of Multiple Endocrine Neoplasia Type 2
Clin. Chem., March 1, 2004; 50(3): 522 - 529.
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S. Haglund, M. Lindqvist, S. Almer, C. Peterson, and J. Taipalensuu
Pyrosequencing of TPMT Alleles in a General Swedish Population and in Patients with Inflammatory Bowel Disease
Clin. Chem., February 1, 2004; 50(2): 288 - 295.
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