A total of 100 patients in Beijing Cancer Hospital with CRC confirmed by histopathology between October 2008 and August 2009 were investigated for KRAS mutations in our laboratory. Formalin-fixed, paraffin-embedded (FFPE) samples with ≥ 50% tumor cells were collected. An A549 cell line was preserved in our laboratory and normal intestinal mucosa was provided by the tissue bank of our hospital.

Genomic DNA of FFPE sections was extracted using E.Z.N.A.FFPE DNA Kit (Lot. D3399-01, OMEGA, USA) according to the manufacturer’s instructions. Genomic DNAs of A549 and normal intestinal mucosa were extracted using EasyPure Genomic DNA Extraction Kit (Lot. D60916, TransGen Biotech, China) according to the manufacturer’s instructions. All genomic DNAs were stored at -20°C until further research.

The concentrations of DNA from the A549 cell line and normal intestinal mucosa were determined by fluorometry. Serial dilutions were prepared by putting A549 cell DNA into wild-type DNA to produce dilutions with the following contents of mutant DNA: 100%, 50%, 20%, 15%, 10%, 5%, 1%, 0%.

A DNA fragment including exon 2 of the KRAS gene was amplified by PCR using primers (KRAS-F: 5'-GGTACTGGTGGAGTATTTGATAG-3', KRAS-R: 5'-TGGTCCTGCACCAGTAATATG-3') with a product size of 248 bp. Each PCR reaction consisted of 10 × LA PCR buffer II 2 μL, 10 mmol/L dNTPs 2 μL, LA Taq 0.2 μL (DRR200A, TAKARA), genomic DNA 2 μL, 10 μmol/L forward primer 0.5 μL, 10 μmol/L reverse primer 0.5 μL in a final volume of 20 μL. The cycling conditions were 94°C for 5 min, 45 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 20 s, final extension at 72°C for 10 min, and ended at 4°C. The PCR products were determined by 3% agarose gel electrophoresis and then sequenced using the same forward primer by Invitrogen 3730XL genetic analyzer. The sequencing results were analyzed with Chromas software under the condition of signal/noise > 98%.

The KRAS StripAssay kit (Lot. 5-590) was kindly provided by ViennaLab of Austria. All procedures were conducted according to the manufacturer’s instructions. Briefly, PCR products were amplified in a tube containing 15 μL amplification mix, 5 μL diluted Taq DNA polymerase (1 U), 5 μL DNA template (50 ng genome DNA). The cycling conditions were 94°C for 2 min, 35 cycles of 94°C for 60 s, 70°C for 50 s, 56°C for 50 s and 60°C for 60 s, final extension at 6°C for 3 min. PCR products with fragment lengths 151 bp and 204 bp were determined by 3% agarose gel electrophoresis after PCR amplification. Following hybridization (45°C, shaking waterbath), stringent washing (45°C, shaking waterbath) and color development (room temperature), the results were interpreted using the enclosed Collector sheet.

A DNA fragment including codons 12 and 13 of the KRAS gene was amplified by PCR using primers (forward: 5'-biotin-TGACTGAATATAAACTTGTGGTAGTTG-3', reverse: 5'-TCGTCCACAAAATGATTCTGAA-3') with a product size of 91 bp. Each PCR reaction consisted of 10 × PCR buffer 5 μL, 10 mmol/L dNTPs 4 μL, Hotstart Taq 0.4 μL [Gene Tech (Shanghai) Company Limited], genomic DNA 4 μL, 10 mmol/L forward primer 0.5 μL, 10 mmol/L reverse primer 0.5 μL in a final volume of 50 μL. The cycling conditions were 95°C for 3 min, 45 cycles of 95°C for 10 s, 56°C for 20 s and 72°C for 30 s, final extension at 72°C for 5 min. The PCR products were determined by 3% agarose gel electrophoresis and ssDNA was prepared as described[16]. Mutation detection of KRAS codons 12 and 13 by the Pyrosequencing PyroMark™ Q24 system was done following the manufacturer’s instructions (see http://www.pyrosequencing.com/ for more information).

The study included 54 males and 46 females with a median age of 59 years (range 22-82 years). The primary locations of tumors were the colon (n = 58) and rectum (n = 42). The mutation rate in females (about 43%) was slightly higher than that in males (about 30%), and the mutation rate in colon and rectal cancers was similar. All patients had a single mutation site. A total of 6 mutation types were detected in this study: GGT>GAT, GGT>GTT, GTT>GCT, GTT>TGT, GTT>AGT, GGC>GAC (wild-type codon 12: GGT; wild-type codon 13: GGC) (Figure 1). Three main mutations Gly12Asp (GGT>GAT), Gly12Val (GGT>GTT) and Gly13Asp (GGC>GAC) accounted for about 80.0% (28/34) of all mutations.

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Figure 1 Mutation patterns of KRAS codons 12 and 13 by the 3 methods in this study. A: Wild-type codons 12 and 13 with sequence GGT(12)GGC(13); B: Mutant codon 12 with GGT>GAT; C: Mutant codon 12 with GGT>GTT; D: Mutant codon 13 with GGC>GAC; E: Mutant codon 12 with GGT>GCT; F: Mutant codon 12 with GGT>AGT; G: Mutant codon 12 with GGT>TGT.

Serial dilutions with different contents of mutant DNA were detected by the 3 methods. All 3 methods could correctly identify the Gly12Ser mutation in dilutions containing 15% or more mutant DNA. Dideoxy sequencing failed to detect the mutation in dilutions containing 10% or less mutant DNA. Pyrosequencing failed to detect the mutation in dilutions containing 1% mutant DNA, and only the KRAS StripAssay could unambiguously identify 1% mutant DNA in the dilutions. Thus the sensitivity was highest in the KRAS StripAssay (1%), followed by pyrosequencing (5%), while dideoxy sequencing was lowest (15%) (Table 1).

The KRAS mutation was detected in 34/100 (34%) of patients by dideoxy sequencing with 15/54 (27.8%) of males and 19/46 (41.3%) of females, 37/100 (37%) of patients by KRAS StripAssay with 16/54 (29.6%) of males and 21/46 (45.7%) of females, and in 37/100 (37%) of patients by pyrosequencing with 18/54 (33.3%) of males and 19/46 (41.3%) of females. Three main mutations Gly12Asp, Gly12Val and Gly13Asp accounted for 82.4% (28/34), 78.4% (29/37) and 83.8% (31/37) of all mutations by dideoxy sequencing, KRAS StripAssay and pyrosequencing, respectively. The overall results of the 3 methods were similar, with a few discrepancies.

Thirty-three of the 100 patients were identified to have KRAS mutations by all 3 methods, and 8 patients (sample No. 5, 11, 14, 29, 44, 46, 48, 71) showed conflicting results between the 3 methods: 4 mutations (sample No. 5, 44, 46, 48) which were not detected by dideoxy sequencing and the KRAS StripAssay, were identified by pyrosequencing; 3 mutations (sample No. 11, 14, 71) which were not detected by dideoxy sequencing and pyrosequencing were identified by the KRAS StripAssay; one mutation (sample No. 29) not detected by pyrosequencing was idenified by dideoxy sequencing and the KRAS StripAssay (Figure 2). In addition, among these discordant results, the mutations identified by dideoxy sequencing were consistent either with the KRAS StripAssay or with pyrosequencing (Table 2). This indicated that although the sensitivity of dideoxy sequencing was low, its accuracy was high.

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Figure 2 Representative discrepant samples and concordant samples between the 3 methods. A: Mutation of sample 5 was not detected by dideoxy sequencing and the KRAS StripAssay but identified by pyrosequencing; B: Mutation of sample 11 was not detected by dideoxy sequencing and pyrosequencing but identified by the KRAS StripAssay; C: Mutation of sample 29 was not detected by pyrosequencing but identified by dideoxy sequencing and the KRAS StripAssay; D: Mutation of sample 82 was detected by all 3 methods.

At present, more and more colorectal cancer (CRC) patients are treated with cetuximab [a monoclonal antibody of epidermal growth factor receptor (EGFR)]. It had been confirmed that KRAS mutated patients could not benefit from cetuximab, so the US Food and Drug Administration recommended that patients proposed for cetuximab treatment should undergo KRAS mutation analysis.

Besides the traditional methods (e.g. dideoxy sequencing), more and more methods (various kinds of kits) have been developed to detect KRAS mutation. The authors compared methods and demonstrated differences between dideoxy sequencing, the KRAS StripAssay and pyrosequencing, with indications that although dideoxy sequencing is a traditional method, it is still in an invincible position because of its low cost and high accuracy.

Recently, many reports have highlighted the advantages of new methods, and disregarded the traditional methods in the detection of KRAS mutation. This was the first study to compare the differences between dideoxy sequencing, the KRAS StripAssay and pyrosequencing in KRAS detection. The study indicated that although the sensitivity of dideoxy sequencing was lower than the other 2 methods, it was still widely used by many researchers because of its superior accuracy.

The study could help researchers to choose a suitable method for detection of KRAS mutation according to the sample size, equipment platform and economic status, etc. In their opinion, dideoxy sequencing could be easily carried out in any laboratory.

In normal physiological conditions, KRAS is regulated by its upstream protein EGFR and plays an important role in the development and progression of tumors. Cetuximab could inhibit the tumors through blocking EGFR and the downstream signal pathway. If KRAS was mutated, KRAS protein was activated without the regulation of EGFR, so cetuximab could not have an effect.

The paper deals with the comparison of 3 DNA sequencing methods in order to detect KRAS mutations. The authors postulate that dideoxy sequencing and pyrosequencing techniques have already been widely used to detect KRAS mutations but the KRAS StripAssay has not been widely used all over the world. The paper is well written and well documented.