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From the Departments of Pathology
*
and Medicine,
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Brigham and Women’s Hospital, Boston; the Department of Medical Oncology,
Dana-Farber Cancer Institute, Boston; Harvard Medical School,
Boston; Biotage AB and Biosystems,
Foxborough; and the Department of Pathology,
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Massachusetts General Hospital, Boston, Massachusetts
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Pyrosequencing (Biotage AB and Biosystems, Uppsala, Sweden) is real-time, nonelectrophoretic, nucleotide extension sequencing for various applications.1, 2, 3 In Pyrosequencing, pyrophosphate is generated only when a dispensed nucleotide anneals to the template and a primer extends by DNA polymerase. Subsequently, pyrophosphate is converted to fluorescence emission, the intensity of which is proportional to the amounts of annealed and extended nucleotide molecules. The Pyrosequencing assay for single-nucleotide polymorphisms (SNPs) can quantify the relative amount of each allele very accurately.1 It can also be applied to quantitative CpG island methylation analysis.4, 5, 6
In this study, we developed a sensitive sequencing assay for the detection of KRAS mutations using Pyrosequencing technology. Although Pyrosequencing typically reads at most 40 to 50 nucleotides, we demonstrate that it has higher analytical sensitivity for the detection of mutant DNA mixed in wild-type DNA when compared with dideoxy (eg, BigDye Terminator) sequencing. The detection limit of our KRAS Pyrosequencing was approximately 3 to 5% of mutant KRAS allele. Our Pyrosequencing method shows good precision, and its ability to quantify the amount of a mutant allele is very useful for assay validation and quality assurance. Moreover, our method effectively works on paraffin-embedded archival tumor tissue and on DNA samples after whole-genome amplification (WGA). High analytical sensitivity is particularly important to avoid bias caused by different features of tumors, such as tumors with abundant inflammatory cells seen in colon cancer with microsatellite instability, desmoplastic pancreatic cancer with cellular and abundant stroma, or previously treated tumors with only scanty remaining neoplastic cells. Our Pyrosequencing assay is a simple, robust, and high-throughput mutation detection assay that can be applied to large epidemiological studies and clinical trials.
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Materials and Methods |
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WGA, PCR, and Dideoxy (BigDye Terminator) Sequencing
Whole genome amplification (WGA) is a useful technique to preserve original study material for many different assays and for future studies. In WGA, genomic DNA is amplified by PCR using primers consisting of random sequence of 15 nucleotides.8
We have extensively used this technique and have shown that WGA does not significantly affect results of microsatellite analysis or sequencing of KRAS or BRAF by dideoxy sequencing (unpublished data). Each PCR mix contained 20 pmol of the random primers, 1.69 nmol each of dNTP, 2.5 mmol/L MgCl2, 1x PCR buffer (Applied Biosystems, Foster City, CA), 0.5 U of AmpliTaq Gold (Applied Biosystems), and 2 µl of template DNA solution in a total volume of 27 µl. PCR conditions consisted of initial denaturing at 94°C for 1 minute; 50 cycles of 94°C for 30 seconds, 37°C for 2 minutes, 41.5°C for 30 seconds, 46°C for 30 seconds, 50.5°C for 30 seconds, 55°C for 2 minutes, and 68°C for 30 seconds; and final extension at 72°C for 1 minute. PCR Primers for KRAS dideoxy sequencing were as follows: KRAS-F14, forward, 5'-tgt aaa acg acg gcc agt tgt gtg aca tgt tct aat ata gtc ac-3'; and KRAS-R7, reverse, 5'-aga atg gtc ctg cac cag taa-3'. Each PCR mix contained the forward and reverse primers (each 10 pmol), 1.69 nmol each of dNTP, 3 mmol/L MgCl2, 1x PCR buffer, 0.75 U of AmpliTaq Gold, and 2 µl of template WGA product in a total volume of 27 µl. PCR conditions consisted of initial denaturing at 94°C for 1 minute; 50 cycles of 95°C for 20 seconds, 50°C for 20 seconds, and 72°C for 40 seconds; and final extension at 72°C for 1 minute. The PCR products were purified using QIAquik PCR Purification kit (Qiagen). Cycle sequencing was performed using BigDye Terminator kit (Applied Biosystems), and analyzed by ABI 3730 (Applied Biosystems).
Development of KRAS Pyrosequencing Assay (Figure 1)
PCR amplification primers for Pyrosequencing were as follows: KRAS-F, forward, 5'-nnn ggc ctg ctg aaa atg act gaa-3'; and KRAS-R, reverse biotinylated primer, 5'-tta gct gta tcg tca agg cac tct-3'. Each PCR mix contained the forward and reverse primers (each 20 pmol), 2.81 nmol each of dNTP, 3 mmol/L MgCl2, 1x PCR buffer, 1.25 U of AmpliTaq Gold, and 5 µl of template WGA product in a total volume of 50 µl. PCR conditions consisted of initial denaturing at 94°C for 1 minute; 50 cycles of 95°C for 20 seconds, 58°C for 20 seconds, and 72°C for 40 seconds; and final extension at 72°C for 1 minute. The PCR products were electrophoresed in an agarose gel to confirm successful amplification of the 82-bp PCR product. The PCR products (each 10 µl) were sequenced by Pyrosequencing PSQ96 HS System (Biotage AB) following the manufacturer’s instructions, using all three Pyrosequencing primers (see below). Nucleotide dispensation order was cyclic (CTAG from 5' to 3').
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. One can use KRAS-PF2 primer to sequence all nucleotides of KRAS codons 12/13 and beyond; however, we found that the use of KRAS-PF1 and KRAS-PF3 primers increases the sensitivity of detection for specific KRAS mutations (data not shown). Moreover, the use of three different primers serves as a quality control measure because all codon 12/13 mutations, except for those in the first nucleotide of codon 12, can be detected by more than one primer. Pyrograms of wild-type KRAS codons 12 and 13 sequence by these primers are shown in Figure 2 A D and G
. The primer KRAS-PF1 (5'-tgt ggt agt tgg agc tg-3') ends at the first nucleotide of codon 12 to detect mutations in the second nucleotide of codon 12 [c.35G>A (codon 12 GAT), c.35G>T (codon 12 GTT), and c.35G>C (codon 12 GCT)] (Figure 1B)
. Because of cyclic CTAG nucleotide dispensation, all three mutations in the second nucleotide of codon 12 can be detected by dispensation of the first three nucleotides CTA with the highest signal-to-noise ratio (Figure 2 B and C)
. Pyrosequencing using KRAS-PF1 for the c.35G>T (codon 12 GTT) mutation is particularly sensitive, because the c.35G>T mutation (codon 12 GTT) creates a T dinucleotide run so that the first T peak on a Pyrogram by KRAS-PF1 primer is twice as high as a peak for one nucleotide change such as c.35G>A.
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. Because of cyclic CTAG nucleotide dispensation, all three mutations in the first nucleotide of codon 12 can be detected by the first three nucleotides CTA with the highest signal-to-noise ratio (Figure 2 E and F)
. The primer KRAS-PF3 (5'-tgg tag ttg gag ctg gt-3') ends at the third nucleotide of codon 12 to detect all mutations in codon 13 [particularly c.38G>A (codon 13 GAC)] with high sensitivity (Figures 1D
2H)
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Comparison of Analytical Sensitivities of Pyrosequencing and BigDye Terminator Sequencing and Statistical Analysis
For cell line DNA mixing study (Figure 3)
, each mutant cell line DNA sample (SW480 or LoVo) was mixed with a wild-type DNA sample (OVCAR) in dilutions of 50, 30, 20, 10, 5, 3, and 2%. All cell line DNA mixtures were prepared in duplicate. All cell line DNA mixtures were amplified by WGA in triplicate. Subsequently, three aliquots from each triplicate WGA were independently amplified by PCR for KRAS and sequenced by Pyrosequencing and BigDye Terminator methods. In addition, three aliquots from each original DNA mixture without WGA were independently amplified by PCR for KRAS and sequenced by Pyrosequencing and BigDye Terminator methods. Thus, there were a total of 24 [= 2 cell lines x 2 duplicate x (3 + 3) aliquots] independent results by both sequencing methods for each dilution.
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, each mutant DNA was mixed with normal DNA in dilutions of 50, 30, 20, 10, and 5%. From each DNA mixture and from each pancreatic cancer sample with unknown KRAS status, DNA was amplified by WGA in triplicate, and three aliquots (each from triplicate WGA) were subsequently amplified by PCR for KRAS, followed by Pyrosequencing and BigDye sequencing. Thus, for paraffin colon cancer mixing study, there were 15 (= 5 samples with different mutations x 3 aliquots) independent results by both sequencing methods for each dilution. For pancreatic cancers, there were 27 (= 9 tumors x 3 aliquots) independent results. Results of both sequencing methods were compared. Statistical analysis was performed using Fisher’s exact test on categorical data by SAS program (version 8.2; SAS Institute, Cary, NC).
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Results |
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. SDs of the mutant allele quantifications in six repeated runs for each DNA mixing ratio ranged from 0.8 to 3.1%. WGA appeared to decrease the amounts of the mutant alleles detected by Pyrosequencing compared with KRAS Pyrosequencing without WGA, indicating subtle, but consistent bias introduced by WGA. Nonetheless, allele quantifications by Pyrosequencing assay were very precise and reproducible, even when whole-genome-amplified DNA samples were used. Allele quantification by Pyrosequencing allows us to perform quality control and quality assurance easily and objectively.
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shows representative results of cell line mixing study on the 3 and 2% mixes. In the cell line mixing study, there were no or insignificant differences between BigDye sequencing and Pyrosequencing when examining mixtures in which the proportion of mutant DNA was either high or very low (ie, mixtures of 50, 30, and 20% as well as 3 and 2%). However, there were significant differences in detection rates in the 24 runs for the 10 and 5% mutant mixtures (Table 1)
. Particularly, in the 5% mix, BigDye sequencing could detect mutations only in 8 of 24 runs, whereas Pyrosequencing could detect mutations in 23 of 24 runs (P = 0.000008).
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In the paraffin colon cancer mixing study, we repeated WGA reactions in triplicate using each DNA mixture as a template, followed by specific PCR for KRAS. There were no significant differences between BigDye sequencing results and Pyrosequencing results for the 50 and 30% mutant mixtures. However, for both the 20 and 10% mutant mixtures, significant differences were evident (Table 2)
. Figure 6B
shows representative Pyrograms and corresponding BigDye sequencing results on the 20, 10, and 5% mutant mixtures. Pyrosequencing detected mutations in all (100%) of the 15 runs for the 20 and 10% mixtures, whereas BigDye sequencing detected mutations in only 8 (53%) of the 15 runs for the 20% mixture (P = 0.006 for the comparison between Pyrosequencing and BigDye sequencing) and in only 6 (40%) of the 15 runs for the 10% mixture (P = 0.0007). For the 5% mixture, Pyrosequencing could still detect mutations in 10 (67%) of the 15 runs, whereas BigDye sequencing could detect mutations in only 7 (47%) of the 15 runs; however, this difference did not reach the level of statistical significance.
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Discussion |
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WGA procedures have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have optimized our Pyrosequencing methods so that we can use whole-genome-amplified DNA from paraffin-embedded tissue. By allele quantification, we demonstrated that fractions of mutant KRAS slightly decreased in whole-genome-amplified DNA samples, indicating that WGA reactions amplify the KRAS wild-type allele slightly better than the KRAS mutant allele (Figure 5)
. However, allele quantification on whole-genome-amplified DNA samples was still very precise, and mutation detection rates for whole-genome-amplified DNA samples were not significantly different from DNA samples that did not undergo WGA. Pyrosequencing showed superior sensitivities to dideoxy sequencing for both whole-genome-amplified DNA samples and those without WGA, including DNA samples from paraffin-embedded tissue. We have used PCR-based random oligonucleotide primed WGA (also known as primer extension preamplification) described by Zhang et al.12
Dietmaier et al8
showed that this method could effectively be used for paraffin-embedded tissue. Of note, Dean et al13
also described WGA by multiple displacement amplification (MDA) using phi 29 DNA polymerase that works optimally at room temperature and can amplify even high molecular weight DNA.13
WGA by MDA has a potential for use in common clinical molecular assays.14
However, the MDA WGA method failed to efficiently amplify DNA extracted from formalin-fixed paraffin-embedded tissue (unpublished data). Regardless of techniques, the use of WGA is potentially useful in both research and clinical settings, because it spares original samples and because many biomarkers can be tested even when the amounts of samples are limited.
A number of alternative KRAS mutation detection methods have been described in the literature. Similar to Pyrosequencing, single-nucleotide extension methods such as solid-phase minisequencing15, 16, 17 can accurately measure mutant allele relative to wild-type allele. However, the use of radioisotope greatly limits clinical usefulness of solid-phase minisequencing assay. Moreover, the technique is limited to single-nucleotide reading. Pyrosequencing represents a similar nucleotide extension technology with nonradioisotopic fluorescence detection platform that can read up to 40 to 50 nucleotides.
In addition, a variety of nonsequencing methods for KRAS mutation detection have been developed. Such methods include PCR-reverse dot blot,18 allele-specific oligonucleotide hybridization,19 activated RAS-GTP specific biosensor,20 real-time allele specific PCR,21 PCR followed by denaturing HPLC,22 oligonucleotide ligation assay,23 PCR-SSCP,24 PCR-DGGE,25 restriction endonuclease-mediated selective PCR,26 and PCR followed by HPLC-electrospray ionization tandem mass spectrometry.27 Innovative approaches for the detection of somatic mutations in general have been reviewed.28, 29, 30, 31 Although extreme sensitivity can be achieved, a general problem with nonsequencing methods is that it is difficult or impossible to confirm independently the existence of any mutations that are identified.32 In this regard, sequencing methods to determine the exact identity of a specific mutation have a definitive advantage. Compared with nonsequencing methods, sequencing methods such as dideoxy sequencing and Pyrosequencing can serve as confirmatory tests to identify a specific mutation, even a rare variant. In fact, nonsequencing methods may have difficulty in detecting rare mutations, including two mutations in one allele, unless one uses an expanded panel of primers or probes to detect those rare variants. Furthermore, sequencing methods can demonstrate DNA sequence around specific nucleotide(s) of interest, providing a quality assurance measure that is especially important in clinical settings.
Digital PCR described by Vogelstein and Kinzler32 does offer the capability of quantifying mutant allele very accurately. However, its application to a large-scale study is currently limited because of the cost and labor-intensive nature of this technology. Alternatively, the array-based KRAS mutation detection system described by Prix et al33 uses peptide nucleic acid-mediated PCR clamping followed by biochip array hybridization. Nonetheless, this method may fail to detect some rare mutations. Moreover, the applicability of this technique to paraffin-embedded tissue and its cost effectiveness and limited throughput remain important issues.
One considerable advantage of Pyrosequencing is its ability to accurately quantify the amount of each allele. This is particularly useful in assay validation, quality control, and quality assurance, as we validated our KRAS Pyrosequencing by demonstrating good assay precision and reproducibility in quantifying the KRAS mutant allele in each one of the DNA mixtures. Allele quantification by Pyrosequencing allows objective quality control and quality assurance, and assay performance can be easily monitored by appropriate controls.
Another advantage of Pyrosequencing is that it can read a nucleotide sequence starting from the first nucleotide right next to a Pyrosequencing primer, so that one can design relatively small PCR products. Designing smaller PCR products is especially useful for degraded DNA samples, particularly DNA derived from paraffin-embedded tissue, in which DNA is typically fragmented into short fragments.
Compared with dideoxy sequencing, we believe that the Pyrosequencing assay offers simplicity and cost effectiveness, particularly in the setting of large-scale projects and clinical assays. Whereas dideoxy (BigDye Terminator) sequencing requires purification of PCR products, there is no need for a separate purification step with Pyrosequencing after PCR products are obtained. Thus, KRAS Pyrosequencing may be more cost effective than dideoxy (BigDye Terminator) sequencing in terms of both reagents and labor time.
As an alternative to Pyrosequencing, laser capture microdissection technique can collect pure population of tumor cells to increase sensitivity for DNA sequencing. However, performing laser capture microdissection is time consuming and labor intensive and yields less of DNA than manual tissue dissection, thereby limiting a menu of biomarkers that can be investigated.
Previous Pyrosequencing methods for NRAS mutation detection showed a detection limit of 15% of mutation-to-wild type ratio (ie, 
13% of mutant allele among total alleles).34
This detection limit cannot be simply compared with that of our KRAS Pyrosequencing, because the detection limit is influenced by many different factors including specimens, the sequence to be analyzed (eg, KRAS vs. NRAS), primer design, the number of PCR cycles, and PCR bias. In fact, we showed evidence of a slight WGA PCR bias in favor of the wild-type KRAS allele. Consistent PCR bias may have some impact on gene copy number determinations in quantitative molecular genetic assays.35
Nonetheless, because of our carefully designed Pyrosequencing primer set, our KRAS Pyrosequencing assay achieves remarkable sensitivity of detecting approximately 5% of mutant KRAS alleles. Our data indicate that Pyrosequencing offers a superior analytical sensitivity when compared with dideoxy (BigDye Terminator) sequencing.
In conclusion, we developed a simple, robust, and sensitive sequencing assay for KRAS mutation detection using high-throughput Pyrosequencing technology. Our assay can be used for paraffin-embedded archival tissue in clinical settings and large population-based studies. The use of WGA can significantly limit the amounts of DNA required for analysis and allows many biomarkers to be tested on the same sample set.
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Footnotes |
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Supported by National Institutes of Health grants P01-9483703, P01-9467802, and R01-9485602 and by Biotage AB and Biosystems.
Accepted for publication April 19, 2005.
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S. Ogino, K. Nosho, J. A. Meyerhardt, G. J. Kirkner, A. T. Chan, T. Kawasaki, E. L. Giovannucci, M. Loda, and C. S. Fuchs Cohort Study of Fatty Acid Synthase Expression and Patient Survival in Colon Cancer J. Clin. Oncol., December 10, 2008; 26(35): 5713 - 5720. [Abstract] [Full Text] [PDF]
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 S. Ogino, K. Nosho, G. J. Kirkner, T. Kawasaki, A. T. Chan, E. S. Schernhammer, E. L. Giovannucci, and C. S. Fuchs A Cohort Study of Tumoral LINE-1 Hypomethylation and Prognosis in Colon Cancer J Natl Cancer Inst, December 3, 2008; 100(23): 1734 - 1738. [Abstract] [Full Text] [PDF]
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I. Fichtner, J. Rolff, R. Soong, J. Hoffmann, S. Hammer, A. Sommer, M. Becker, and J. Merk Establishment of Patient-Derived Non-Small Cell Lung Cancer Xenografts as Models for the Identification of Predictive Biomarkers Clin. Cancer Res., October 15, 2008; 14(20): 6456 - 6468. [Abstract] [Full Text] [PDF]
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E. S. Schernhammer, E. Giovannuccci, C. S. Fuchs, and S. Ogino A Prospective Study of Dietary Folate and Vitamin B and Colon Cancer According to Microsatellite Instability and KRAS Mutational Status Cancer Epidemiol. Biomarkers Prev., October 1, 2008; 17(10): 2895 - 2898. [Abstract] [Full Text] [PDF]
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D. T. Croft Jr, R. M. Jordan, H. L. Patney, C. D. Shriver, M. N. Vernalis, T. J. Orchard, and D. L. Ellsworth Performance of Whole-Genome Amplified DNA Isolated from Serum and Plasma on High-Density Single Nucleotide Polymorphism Arrays J. Mol. Diagn., May 1, 2008; 10(3): 249 - 257. [Abstract] [Full Text] [PDF]
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 N. S. Butler, A. Theodossis, A. I. Webb, M. A. Dunstone, R. Nastovska, S. H. Ramarathinam, J. Rossjohn, A. W. Purcell, and S. Perlman Structural and Biological Basis of CTL Escape in Coronavirus-Infected Mice J. Immunol., March 15, 2008; 180(6): 3926 - 3937. [Abstract] [Full Text] [PDF]
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S. Ogino and A. Goel Molecular Classification and Correlates in Colorectal Cancer J. Mol. Diagn., January 1, 2008; 10(1): 13 - 27. [Abstract] [Full Text] [PDF]
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S. Ogino, T. Kawasaki, G. J Kirkner, Y. Suemoto, J. A Meyerhardt, and C. S Fuchs Molecular correlates with MGMT promoter methylation and silencing support CpG island methylator phenotype-low (CIMP-low) in colorectal cancer Gut, November 1, 2007; 56(11): 1564 - 1571. [Abstract] [Full Text] [PDF]
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C. Spittle, M. R. Ward, K. L. Nathanson, P. A. Gimotty, E. Rappaport, M. S. Brose, A. Medina, R. Letrero, M. Herlyn, and R. H. Edwards Application of a BRAF Pyrosequencing Assay for Mutation Detection and Copy Number Analysis in Malignant Melanoma J. Mol. Diagn., September 1, 2007; 9(4): 464 - 471. [Abstract] [Full Text] [PDF]
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E. Dejeux, V. Audard, C. Cavard, I. G. Gut, B. Terris, and J. Tost Rapid Identification of Promoter Hypermethylation in Hepatocellular Carcinoma by Pyrosequencing of Etiologically Homogeneous Sample Pools J. Mol. Diagn., September 1, 2007; 9(4): 510 - 520. [Abstract] [Full Text] [PDF]
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 S. Ogino, A. Hazra, G. J. Tranah, G. J. Kirkner, T. Kawasaki, K. Nosho, M. Ohnishi, Y. Suemoto, J. A. Meyerhardt, D. J. Hunter, et al. MGMT germline polymorphism is associated with somatic MGMT promoter methylation and gene silencing in colorectal cancer Carcinogenesis, September 1, 2007; 28(9): 1985 - 1990. [Abstract] [Full Text] [PDF]
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S. Ogino, T. Kawasaki, G. J. Kirkner, P. Kraft, M. Loda, and C. S. Fuchs Evaluation of Markers for CpG Island Methylator Phenotype (CIMP) in Colorectal Cancer by a Large Population-Based Sample J. Mol. Diagn., July 1, 2007; 9(3): 305 - 314. [Abstract] [Full Text] [PDF]
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S. Ogino, T. Kawasaki, G. J. Kirkner, M. Loda, and C. S. Fuchs CpG Island Methylator Phenotype-Low (CIMP-Low) in Colorectal Cancer: Possible Associations with Male Sex and KRAS Mutations J. Mol. Diagn., November 1, 2006; 8(5): 582 - 588. [Abstract] [Full Text] [PDF]
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S Ogino, M Cantor, T Kawasaki, M Brahmandam, G J Kirkner, D J Weisenberger, M Campan, P W Laird, M Loda, and C S Fuchs CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies Gut, July 1, 2006; 55(7): 1000 - 1006. [Abstract] [Full Text] [PDF]
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S. Ogino, J. A. Meyerhardt, M. Cantor, M. Brahmandam, J. W. Clark, C. Namgyal, T. Kawasaki, K. Kinsella, A. L. Michelini, P. C. Enzinger, et al. Molecular Alterations in Tumors and Response to Combination Chemotherapy with Gefitinib for Advanced Colorectal Cancer Clin. Cancer Res., September 15, 2005; 11(18): 6650 - 6656. [Abstract] [Full Text] [PDF]
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