CLB-08843; No. of pages: 6; 4C: Clinical Biochemistry xxx (2014) xxx–xxx
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Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique Feifei Xie a,1, Jie Huang b,1, Shoufang Qu b,1, Weili Wu a, Jun Jiang a, Huagui Wang a, Shujuan Wang a, Qi Liu c, Senlin Zhang c, Lizhi Xu c, Shangxian Gao b, Yunqing Zhang d, Jinyin Zhao a,⁎, Weijun Chen a,⁎ a
Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China Division of In Vitro Diagnostic Reagents, National Institutes for Food and Drug Control (NIFDC), Beijing 100050, China Beijing Macro & Micro Test Biotech Company, Beijing 101312, China d Department of Dermatology, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou, Guang Dong Province 510630, China b c
a r t i c l e
i n f o
Article history: Received 24 February 2014 Received in revised form 15 July 2014 Accepted 20 August 2014 Available online xxxx Keywords: KRAS mutation detection Low-level mutation Colorectal cancer
a b s t r a c t Objectives: The objective of this study is to develop a novel and sensitive method for KRAS codon 12 mutation testing. Design and methods: We developed a sensitive one-step real-time digestion-and-block TaqMan probe PCR (RTDB-PCR) technique that uses a thermostable endonuclease and a minor groove binder (MGB) blocker to detect KRAS codon 12 mutations. Dilution mimic DNA panels were used to assess the sensitivity of this technique. The RTDB-PCR method was performed and compared with three other methods: PCR sequencing, mutantenriched PCR sequencing and mutant-enriched PCR-MassArray. A total of 100 formalin-fixed paraffinembedded (FFPE) metastatic colorectal cancer (mCRC) specimens were also tested by all four methods. Results: The RTDB-PCR was sensitive to as little as 0.01% mutant DNA, significantly higher than other methods. Among the 100 FFPE mCRC specimens examined, 45 tested positive for KRAS codon 12 mutations according to RTDB-PCR, 44 tested positive according to mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, and only 26 samples tested positive according to PCR sequencing. Conclusions: Compared with mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, RTDB-PCR is more cost effective, saves time, and is easier to use, making it suitable for the detection of lowlevel KRAS mutations in the clinic. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction KRAS mutations are frequently found in colorectal cancer (CRC) (39%–43%) [1,2]. The monoclonal antibodies (mAbs) targeting EGFR (e.g., cetuximab and panitumumab) have shown remarkable efficacy in treating cases of mCRC in which the KRAS status is wild-type [3,4]. The American Society for Clinical Oncology (ASCO) and National Comprehensive Cancer Network (NCCN) guidelines both recommended that all mCRC tumors should be tested for KRAS mutations before beginning anti-EGFR mAb therapy [5,6]. The most frequent KRAS alterations associated with mCRC are detected in codons 12 and 13, and six codon 12 mutations account for more than 80% of the mCRC-associated KRAS mutations [7]. Thus, it is imperative to detect KRAS codon 12 mutations in a correct and timely fashion. However, clinical tumor samples are typically composed of both wild-type and mutant DNA, with the proportion of wild-type DNA often vastly exceeding that of the mutant. The PCR ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Zhao), [email protected] (W. Chen). 1 These authors contributed equally to this article.
sequencing method, which is generally considered a gold standard for clinical diagnosis, is reliable only when the mutant-to-wild ratio reaches 10%–20% [8,9]. Thus, more optimal KRAS mutation-testing methods are urgently needed to improve clinical diagnosis. To detect low-level mutations in tumors (e.g., 10−3 to 10−6 mutant to wild-type DNA), a method must have both high selectivity and the ability to enrich minority alleles. Over the past two decades, most of the research on enhancing the detection of minority (mutant) alleles in clinical samples has focused on improving the selectivity of PCRbased technologies and/or enriching the mutant sequences. A number of approaches have been developed, including amplification refractory mutation system (ARMS) [10,11], coamplification at lower denaturation temperature PCR (COLD-PCR) [12–14], locked nucleic acid/peptide nucleic acid clamp PCR (LNA/PNA-PCR) [15,16], mutant-enriched PCR [17,18], one-step enzyme-enriched ARMS PCR [19] and restriction endonuclease-mediated real-time digestion-PCR (RTD-PCR) [19]. Most of these assays were reported to show moderate to high selectivity. However, we do not yet have a universally accepted approach that can be used as a routine diagnostic tool, because it is difficult to achieve high selectivity and enrichment while maintaining accuracy, convenience, and low cost.
http://dx.doi.org/10.1016/j.clinbiochem.2014.08.015 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Xie F, et al, Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.015
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F. Xie et al. / Clinical Biochemistry xxx (2014) xxx–xxx
Here, we developed a one-step assay called real-time digestion-andblock TaqMan probe PCR (RTDB-PCR) for the detection of low-level KRAS codon 12 mutations within large excesses of wild-type KRAS sequences. The highlight of this technique lies in the use of PspGI, a thermostable endonuclease, and a MGB blocker. The wild-type DNA could be digested by PspGI under 75 °C, which made amplification and restriction digestion happen in one reaction. With the help of the MGB blocker, which completely matched the wild-type template, the specificity was further improved. We evaluated the feasibility of using RTDB-PCR on clinical tumor samples and compared it to the use of PCR sequencing, mutant-enriched PCR sequencing and mutantenriched PCR-MassArray. Materials and methods Ethics statement This research was approved by the Review Board of the Beijing Institute of Genomics, the Review Board of the Beijing Genomics Institute, and the Review Board of the National Institutes for Food and Drug Control of China. All participants gave written informed consent for use of their samples in research. Clinical samples A total of 100 tumor samples surgically resected from mCRC patients at the Peking Union Hospital (Beijing, China) were used in this study. Hematoxylin and eosin stained sections of formalin-fixed, paraffinembedded (FFPE) tissue from each sample were reviewed and the areas of highest tumor density (at least 50% tumor cells content) were identified and encircled. For each tumor, the FFPE block with the maximum number of tumor-rich areas was selected and sliced into two 5-μm-thick sections. A total of 60 blood samples from normal healthy controls were collected in EDTA-containing vacuum tubes at the Peking Union Hospital (Beijing, China); these were used to determinate the ΔCt cut-off value. Dilution panels and sensitivity evaluation
using a NanoDrop 8000 (Thermo, Germany) and ranged from 2 ng/μl to 100 ng/μl. Mutation testing methods RTDB-PCR A 141-bp product spanning codon 12 of KRAS exon 2 was amplified by primer F 5′-TAAACTTGTGGTAGTTGGACCT-3′ (forward, mismatched base is bolded and underlined) and primer R 5′-GGTCCTGCACCAGTAA TATGC-3′ (reverse). Thermostable PspGI was used to digest the amplified wild-type products. The MGB blocker (5′-TGGAGCTGGTGGCGT AGG-MGB-3′), which completely matched the wild-type template, was used to further improve the specificity. Signal generation was monitored in real-time using a TaqMan probe (5′-FAM-AGAGTGCCTT GACGATACAGCTAATTCA-BHQ1-3′). The reaction mixture consisted of 12.5 μl of 2 × Premix Ex Taq (TaKaRa), 5 U of PspGI (New England Biolabs, USA), 0.24 μM each of primers F and R, 0.4 μM of MGB blocker, 0.16 μM of TaqMan probe, 0.4 μl of 50 × ROX, 3 μl of templates, and ddH2O up to 25 μl. PCR was performed on an ABI 7300 real-time PCR instrument (Applied Biosystems, USA) according to the following protocol: 3 min of denaturation at 95 °C, followed by 40 cycles of 92 °C for 10 s, 55 °C for 35 s for primer annealing, extension and fluorescence data collection, and 75 °C for 60 s for PspGI cleavage. Each assay was performed in triplicate. The core of this technique is the use of the thermostable endonuclease, PspGI, and an MGB blocker. PspGI, which is purified from Pyrococcus sp. strain GI-H, is an extremely thermostable restriction endonuclease that has a half-life of 2 h at 95 °C. DNA containing the sequence 5′-CCWGG-3′ (W = A or T) is cleaved effectively by PspGI across an optimum temperature range of 75 °C to 85 °C [21]. In our strategy, a mismatched base was introduced into KRAS codon 11 (GCT NCCT) with primer F; once amplified, most of the wild-type KRAS (KRAS-wt) sequences (GCTGGTNCCTGGT) are cleaved at 75 °C for 1 min, enriching the mix for the mutant KRAS sequences (GCTGGT NCCTHHT H ≠ G). A blocker with an MGB label at the 3′ terminus was used to further improve the specificity; it was completely complementary to KRAS-wt and inhibited the amplification of wild-type (but not mutant) sequences. The principle of this design is depicted in Fig. 1.
DNA extraction
Determination of the ΔCt cut-off value To determine the specificity of our RTDB-PCR technique, we performed RTDB-PCR and control assays using 60 human genomic DNA samples from normal healthy controls with concentrations ranging from 2 ng/μl to 100 ng/μl. Reactions lacking PspGI and the MGB blocker were defined as control assays. All samples were assessed in three technical replicates. The ΔCt cut-off value was calculated by determining the average ΔCt between mutant-detecting and control assays and then subtracting a value of 3 times the standard deviation. This was done to ensure that there was a significant difference between true amplification of a mutant allele and non-specific, off-target amplification (according to the protocol provided with the ABI TaqMan® Mutation Detection Assay). The utilized formula was as follows: ΔCt cut-off = Average ΔCt-3 × the standard deviation and ΔCt = Ct (mutant assay) − Ct (control assay). If the ΔCt was below the ΔCt cut-off value, the sample was classified as positive. If the ΔCt was greater than the ΔCt cut-off value, the sample was classified as negative or beyond the limit of detection.
Tumor DNA was extracted from two sections of each sample using a QIAamp DNA FFPE Tissue kit (Qiagen, Germany) according to the manufacturer's instructions. DNA was eluted from the columns with 100 μl of water containing 20 μg/ml RNase A. After incubation at 37 °C for 15 min, the DNA was immediately stored at −20 °C. Normal genomic DNA was extracted from 200 μl of each blood sample using a TIANamp Blood DNA kit (Tiangen, China) according to the manufacturer's instructions. The concentrations of DNA were measured
PCR sequencing. PCR was performed in a 30-μl mixture containing 15 μl of 2× GC buffer (TaKaRa, Japan), 0.25 μΜ each of primers F and R, 1.5 U of rTaq, 4.8 μl of dNTP mixture (2.5 mM), 3 μl of template, and ddH2O up to 30 μl. PCR was performed on a 2720 PCR cycler (Applied Biosystems, USA) according to the following protocol: 3 min of denaturation at 95 °C, followed by 40 cycles of 92 °C for 10 s, 55 °C for 35 s and 72 °C for 60 s. The products were further sequenced using an ABI PRISM 3730xl system (Applied Biosystems, USA).
Recombinants containing the six most common mCRC-related KRAS codon 12 mutations and wild-type KRAS codon 12 were constructed using overlap extension PCR [20] and cloned into the pMD19-T plasmid (TaKaRa, Japan). Recombinants were extracted using TIANprep Mini Plasmid kits (Tiangen, China), and their sequences were confirmed by Sanger sequencing. All primers used in recombinant construction are listed in Supplementary Table 1. The concentration of recombinants was measured using a NanoDrop 8000 (Thermo, Germany). The mutant plasmid DNA was serially mixed with 15,000 copies/μl of wild-type plasmid DNA in the following percentages: 100%, 10%, 1%, 0.1%, 0.01%, and 0%, namely, mutant DNA content was 15,000 copies/μl, 1500 copies/μl, 150 copies/μl, 15 copies/μl of 1.5 copies/μl respectively. It was also serially diluted with 200 ng/μl human genomic DNA (SigmaAldrich, Cat. HRC1; 60,000 copies/μl) in the following percentages: 100%, 10%, 1%, 0.1%, 0.01%, and 0%. These two panels were used for sensitivity determination.
Please cite this article as: Xie F, et al, Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.015
F. Xie et al. / Clinical Biochemistry xxx (2014) xxx–xxx
5’ 3’
CCT CGANNA
5’
B
A Artificial introduction of restriction site 5’ 3’
CCTGGT
No introduction 3’ 5’
CCTGGT
No amplification
5’ 3’
5’ 3’
CCTGAT
3’ G12D 5’
5’ 3’
CCTGCT
3’ G12A 5’
5’ 3’
CCTGTT
3’ 5’ G12V
PO4 5’ 3’ 5’ 5’ 3’
CCTAGT
3’ G12S 5’
CCTCGT
3’ 5’ G12R
5’ 3’
CCTTGT
3’ 5’ G12C
3’ 5’
GCTGGT
No cleavage But blocked
Once amplified PspGI cleavage 5’ 3’
3
3’ 5’
MGB 3’
GCTGGT CGACCA
No amplification
All six mutants amplified with no difference Fig. 1. PspGI and MGB blocker-mediated KRAS mutation assay. (A) A mutagenic primer introduces a variation (denoted in red) that creates a recognition site for PspGI in the wild-type codon 12 target sequence, thereby suppressing the exponential amplification of the modified wild-type alleles. Meanwhile, the amplification of unmodified wild-type alleles is suppressed by a fully complementary MGB blocker. (B) The mutant alleles escape digestion and are exponentially amplified. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Mutant-enriched PCR sequencing. The reaction mixture was the same as described for RTDB-PCR, except that it lacked the MGB blocker and Taqman probe. The initial PCR conditions were as described for RTDBPCR. 5 μl PCR products were taken out for next Mutant-enriched PCRMassArray assay (Sequenom, USA) and the remaining 20 μl products were further sequenced using an ABI PRISM 3730xl system (Applied Biosystems, USA).
Mutant-enriched PCR-MassArray. For our mutant-enriched PCRMassArray assays, two separate well were used to assess the genotype of KRAS codon 12. Primers UEP1 5′-ACTCTTGCCTACGCCAC-3′ and UEP2 5′-CACTCTTGCCTACGCCA-3′ were designed as single-base extension primers for c.34G and c.35G, respectively. Mutant-enriched PCR products (5 μl; described above) were used as the template in order to minimize error between reactions. The unincorporated dNTPs were dephosphorylated by incubation at 37 °C for 40 min with 0.3 U of shrimp alkaline phosphatase (SAP), 0.17 μl of SAP buffer and 1.53 μl of ddH2O. Then, extension reactions were performed in a 9-μl system, as described in the Application Guide (Sequenom, USA). SAP and extension were run on a Veriti 384-well thermal cycler (Applied Biosystems, USA). Three replicate experiments were performed for each sample.
Statistical analysis To evaluate the performance of RTDB-PCR, the kappa values between RTDB-PCR and the other three methods were analyzed. Agreement between the pairs of tests was assessed using Kappa values, with values in the range of 0.00 to 0.20 indicating poor agreement, 0.21 to 0.40 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.80 good agreement, and 0.81 to 1.00 excellent agreement. All statistical calculations were performed with the Statistical Package for the Social Sciences (SPSS) software (version 16.0, SPSS Inc., Brussels, Belgium).
Results Patient and characteristics Of the tumors, 75% (75/100) were located in the colon, and the rest were located in the rectum. The average age of the overall population was 62 ± 12 (range, 28 to 89 years). The grade of differentiation of all 60 samples was documented. Further details on these patients are given in Table 1. Determination of the ΔCt cut-off value To calculate the ΔCt cut-off value for our RTDB-PCR, we tested 60 blood samples collected from healthy people at the Peking Union Hospital. All of the amplifications of these control assays were successful and their Ct values range from 20.18 to 27.86. As to mutant assays, wild-type DNA was digested completely by PspGI, so no detection signal
Table 1 Clinical characteristics of the patients.
Patient characteristics (+)a Sex Male (+) Female (+) Median age (range)b Grade of differentiation Poorly (+) Moderately (+) Well (+) Lymph node metastasis Yes (+) No (+)
Colon cancer
Rectal cancer
Total
75(34)
25(11)
100(45)
45(20) 30(14) 61 ± 13(28–89)
13(5) 12(6) 63 ± 10(44–79)
58(25) 42(20) 62 ± 12(28–89)
10(6) 23(9) 42(19)
2(1) 10(3) 13(7)
12(7) 33(12) 55(26)
33(17) 42(17)
13(6) 12(5)
46(23) 54(22)
Note: a Represents the number of RTDB-PCR positive samples. b Represents the range of patient's age.
Please cite this article as: Xie F, et al, Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.015
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F. Xie et al. / Clinical Biochemistry xxx (2014) xxx–xxx
was collected. Then we defined 40 as the Ct of mutant assay and calculated the ΔCt cut-off value as following: ΔCt cut‐off ¼ Average ΔCt−3 the standard deviation ¼ 16:5−3 1:63 ¼ 11:6:
as calculated by the peak area ratio of mutant to wild-type in the mutant-enriched PCR-MassArray assay (Supplementary Table 2). A sensitivity comparison of these four methods against the 15,000 copies/μl of wild-type plasmid background was depicted in Fig. 2.
Mutation testing of clinical specimens Sensitivity comparison Only RTDB-PCR was sensitive to 0.01% mutant DNA in both backgrounds (15,000 copies/μl of wild type plasmid DNA or 200 ng/μl human genomic DNA). The sensitivity of the mutant-enriched PCRMassArray reached 0.01% in the 200 ng/μl DNA panel, but was only 0.1% in the 15,000 copies/μl of DNA panel. The sensitivity of the mutant-enriched PCR sequencing was 0.1% in both panels, while that of PCR sequencing was only 10% in both panels. After PspGI digestion, the fold of mutant enrichment was as high as 2975 (29.75% to 0.01%),
Comparison between RTDB-PCR and PCR sequencing All 100 of the mCRC FFPE samples received from Peking Union Hospital were successfully tested by RTDB-PCR, and KRAS codon 12 mutations were found in 45 samples. In contrast, only 26 mutations were identified by PCR sequencing. The positive agreement, negative agreement and overall agreement between RTDB-PCR and PCR sequencing were 100%, 74.4% and 81%, respectively, and the kappa value between the two methods was 0.601 (Table 2). We did a simple classification of 45 RTDB-PCR positive samples and found that 34 were
PCR sequencing
RTDB-PCR 100%
IC
IC 0.1%
1%
10%
0.01%
IC
IC
100%
10%
1%
0.1%
0.01%
0%
IC 0%
IC
A
B
Mutant-enriched PCR-MassArray
Mutant-enriched PCR sequencing 100%
10%
1%
W
M
100%
0.1%
0.01%
0%
W
C
M
W
10%
M
0.1%
failure
W
W
M
1%
M
0.01%
M
W
0%
failure
D
Fig. 2. Sensitivity evaluation of RTDB-PCR, PCR sequencing, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray. (A) to (D) respectively described the KRAS G12D sensitivity detection plot of RTDB-PCR, PCR sequencing, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray in a background of 15,000 copies/μl of wild-type plasmids. The proportion of mutant to wild plasmid ranged from 100% to 0%.(A) IC indicated the control assay without PspGI or the MGB blocker. The curves indicated by a red arrow represent the amplification plot of RTDB-PCR detection and 0.01% mutants could be detected by RTDB-PCR. (B) and (C) showed the results of PCR sequencing and mutant-enriched PCR sequencing. Mutant peak was indicated by a red arrow. The sensitivity of the mutant-enriched PCR sequencing was 0.1%, while that of PCR sequencing was only 10%. (D) The extension production peak of mutant-type and wild-type was marked with M and W. The sensitivity of the mutant-enriched PCR-MassArray reached 0.1% in the 15,000 copies/μl of DNA background.
Please cite this article as: Xie F, et al, Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.015
F. Xie et al. / Clinical Biochemistry xxx (2014) xxx–xxx Table 2 Comparison of RTDB-PCR and PCR sequencing. N = 100
PCR sequencing
RTDB-PCR + − Total
+
−
Total
26 0 26
19 55 74
45 55 100
colon cancer and 11 were rectal cancer. More details of the classification of RTDB-PCR positive samples were shown in Table 1. Comparison of RTDB-PCR, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray RTDB-PCR, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, which all use thermostable PspGI to improve their selectivity, successfully identified 45, 44 and 44 samples, respectively. There were one sample tested positive to RTDB-PCR but it has a ΔCt value with 10.68. It suggested that it has very low mutations. The positive agreement, negative agreement and overall agreement between RTDB-PCR and either of the other two methods were 100%, 98.2%, 99%, respectively, and the kappa value in each case was 0.980 (Table 3). 44 of 45 mutant specimens detected by RTDB-PCR were also genotyped based on the sequencing data obtained from the mutantenriched PCR MassArray and mutant-enriched PCR sequencing methods. Among these 44 cases, there were 36 single-base mutations and 8 complex mutations, as follows: 14 G12D (GGT NGAT), 11 G12A (GGT N GCT), 7 G12V (GGT N GTT), 3 G12C (GGT NTGT), 1 G12S (GGT AGT), 4 G12D/G12A (GGTNGAT/GCT), 1 G12V/G12A (GGTNGTT/GCT), 1 G12C/G12A (GGTNTGT/GCT), 1 G12D/G12 C(GGTN GAT/TGT), and 1 G12D/G12A/G12V (GGTNGAT/GCT/GTT). Discussion In the wake of the development of molecular targeted therapy, it has become increasingly important to detect biomarkers quickly, sensitively and accurately [22]. For mutation detection, Sanger sequencing has been widely utilized and still serves as a gold standard in most clinical laboratories, but lower sensitivity limited its utility [23,24]. It is very difficult to obtain homogeneous tumor samples in the clinical setting. Even if we could obtain homogeneous tumor samples, the somatic mutations were also heterogeneous [25]. Mutation content was often so little that the limit of detection of PCR sequencing couldn't reach. Novel diagnostic techniques that purportedly have high selectivity are constantly springing up. However, we do not yet have a uniform standard diagnostic method for detecting mutations, and most of the “highly sensitive” methods that have been reported are laborious, time-consuming, expensive, and/or limited by issues with low template concentrations [26]. In the present study, our RTDB-PCR had higher sensitivities and was able to detect as few as 0.01% mutants in a background of 200 ng wildtype DNA. It is a huge improvement over the sensitivity of PCR sequencing which could detect as few as 10% mutants. The agreement between RTDB-PCR and PCR sequencing for 100 mCRC sample detection was
Table 3 Comparison of RTDB-PCR and the other two enriched methods. N = 100
RTDB-PCR + − Total
Mutant-enriched PCR sequencing
Mutant-enriched PCR-MassArray
+
−
+
−
44 0 43
1 55 56
44 0 43
1 55 56
Total
45 55 100
5
poor (the kappa value was only 0.601), mainly because of the low sensitivity of PCR sequencing. Compared with mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, RTDB-PCR has an equal or slight higher sensitivity. Mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray both detect 44 positive samples, while RTDB-PCR tested one more positive sample (control Ct = 26.16, detection Ct = 36.84, ΔCt = 10.68). In sensitivity assays, we found mutant-enriched PCR sequencing and mutant-enriched PCRMassArray with only 0.1% sensitivity in the 15,000 copies/μl of DNA panel. The ΔCt value of this sample (contains about 5000 copies/μl of total DNA) was greater than the ΔCt value (ΔCt values approximately equal 10) of 0.1% sensitivity, leading to a negative (failed testing) result. As a high-throughput and cost effective method, MassArray iPLEX assay can analyze up to 384 samples with one multiplex assay or up to 384 different multiplexed assays with one sample (see MassARRAY iPLEX® Gold SNP Genotyping instruction). However, mutant-enriched PCR-MassArray requires a separate pre-PCR because of PspGI digestion, which produces a complex operation. Besides, PCR-MassArray needs a relatively high DNA concentration for spotting, which also limits its application especially in low level mutation testing. Mutant-enriched PCR-MassArray and mutant-enriched PCR sequencing could discriminate the specific genotypes of KRAS mutation. After a comprehensive analysis, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray are less suitable for clinical testing because: 1) no evidence showed that the specific genotypes of codons 12 and 13 have different contributions to anti-EGFR mAb therapy; 2) it only include a closed-tube detection and have less contamination risks than mutantenriched PCR sequencing and mutant-enriched PCR-MassArray; 3) it would only take about 2 h whereas mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray would take more than 24 h to finish the tests; and 4) mutant-enriched PCR sequencing and mutantenriched PCR-MassArray both need expensive instruments which cost more than 300,000 USD. Therefore, our novel RTDB-PCR method seems to be a powerful new tool for mutation screening early during disease progression, and thus could have important therapeutic implications. Conflict of interest The authors declare no conflict of interest. Acknowledgments We are grateful to the Peking Union Hospital for providing tissue samples. This project was supported by the Major State Basic Research Development Program of China (grant No. 2010CB529101, 973 Program) and the Chinese 863 Program (2012AA02A201). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.clinbiochem.2014.08.015. References [1] Maughan TS, Adams RA, Smith CG, Meade AM, Seymour MT, Wilson RH, et al. Addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial. Lancet 2011;377:2103–14. [2] Tveit KM, Guren T, Glimelius B, Pfeiffer P, Sorbye H, Pyrhonen S, et al. Phase III trial of cetuximab with continuous or intermittent fluorouracil, leucovorin, and oxaliplatin (Nordic FLOX) versus FLOX alone in first-line treatment of metastatic colorectal cancer: the NORDIC-VII study. J Clin Oncol 2012;30:1755–62. [3] Lievre A, Bachet JB, Le Corre D, Boige V, Landi B, Emile JF, et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res 2006; 66:3992–5. [4] Van Cutsem E, Kohne CH, Hitre E, Zaluski J, Chang Chien CR, Makhson A, et al. Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med 2009;360:1408–17.
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[16] Arcila M, Lau C, Nafa K, Ladanyi M. Detection of KRAS and BRAF mutations in colorectal carcinoma roles for high-sensitivity locked nucleic acid-PCR sequencing and broad-spectrum mass spectrometry genotyping. J Mol Diagn 2011;13:64–73. [17] Asano H, Toyooka S, Tokumo M, Ichimura K, Aoe K, Ito S, et al. Detection of EGFR gene mutation in lung cancer by mutant-enriched polymerase chain reaction assay. Clin Cancer Res 2006;12:43–8. [18] Brevet M, Johnson ML, Azzoli CG, Ladanyi M. Detection of EGFR mutations in plasma DNA from lung cancer patients by mass spectrometry genotyping is predictive of tumor EGFR status and response to EGFR inhibitors. Lung Cancer 2011; 73:96–102. [19] Zhao J, Huang J, Chen Y, Jiang J, Wu W, Wang P, et al. A novel method for detection of mutation in epidermal growth factor receptor. Lung Cancer 2011;74:226–32. [20] Heckman KL, Pease LR. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2007;2:924–32. [21] Morgan R, Xiao J, Xu S. Characterization of an extremely thermostable restriction enzyme, PspGI, from a Pyrococcus strain and cloning of the PspGI restriction–modification system in Escherichia coli. Appl Environ Microbiol 1998;64:3669–73. [22] Endo K, Konishi A, Sasaki H, Takada M, Tanaka H, Okumura M, et al. Epidermal growth factor receptor gene mutation in non-small cell lung cancer using highly sensitive and fast TaqMan PCR assay. Lung Cancer 2005;50:375–84. [23] Kalikaki A, Koutsopoulos A, Hatzidaki D, Trypaki M, Kontopodis E, Stathopoulos E, et al. Clinical outcome of patients with non-small cell lung cancer receiving frontline chemotherapy according to EGFR and K-RAS mutation status. Lung Cancer 2010;69:110–5. [24] Uruga H, Kishi K, Fujii T, Beika Y, Enomoto T, Takaya H, et al. Efficacy of gefitinib for elderly patients with advanced non-small cell lung cancer harboring epidermal growth factor receptor gene mutations: a retrospective analysis. Intern Med 2010; 49:103–7. [25] Zhang X, Zhao Y, Wang M, Yap WS, Chang AY. Detection and comparison of epidermal growth factor receptor mutations in cells and fluid of malignant pleural effusion in non-small cell lung cancer. Lung Cancer 2008;60:175–82. [26] Marchetti A, Gasparini G. K-ras mutations and cetuximab in colorectal cancer. N Engl J Med 2009;360:833–4 [author reply 5-6].
Please cite this article as: Xie F, et al, Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.015
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique Feifei Xie a,1, Jie Huang b,1, Shoufang Qu b,1, Weili Wu a, Jun Jiang a, Huagui Wang a, Shujuan Wang a, Qi Liu c, Senlin Zhang c, Lizhi Xu c, Shangxian Gao b, Yunqing Zhang d, Jinyin Zhao a,⁎, Weijun Chen a,⁎ a
Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China Division of In Vitro Diagnostic Reagents, National Institutes for Food and Drug Control (NIFDC), Beijing 100050, China Beijing Macro & Micro Test Biotech Company, Beijing 101312, China d Department of Dermatology, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou, Guang Dong Province 510630, China b c
a r t i c l e
i n f o
Article history: Received 24 February 2014 Received in revised form 15 July 2014 Accepted 20 August 2014 Available online xxxx Keywords: KRAS mutation detection Low-level mutation Colorectal cancer
a b s t r a c t Objectives: The objective of this study is to develop a novel and sensitive method for KRAS codon 12 mutation testing. Design and methods: We developed a sensitive one-step real-time digestion-and-block TaqMan probe PCR (RTDB-PCR) technique that uses a thermostable endonuclease and a minor groove binder (MGB) blocker to detect KRAS codon 12 mutations. Dilution mimic DNA panels were used to assess the sensitivity of this technique. The RTDB-PCR method was performed and compared with three other methods: PCR sequencing, mutantenriched PCR sequencing and mutant-enriched PCR-MassArray. A total of 100 formalin-fixed paraffinembedded (FFPE) metastatic colorectal cancer (mCRC) specimens were also tested by all four methods. Results: The RTDB-PCR was sensitive to as little as 0.01% mutant DNA, significantly higher than other methods. Among the 100 FFPE mCRC specimens examined, 45 tested positive for KRAS codon 12 mutations according to RTDB-PCR, 44 tested positive according to mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, and only 26 samples tested positive according to PCR sequencing. Conclusions: Compared with mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, RTDB-PCR is more cost effective, saves time, and is easier to use, making it suitable for the detection of lowlevel KRAS mutations in the clinic. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction KRAS mutations are frequently found in colorectal cancer (CRC) (39%–43%) [1,2]. The monoclonal antibodies (mAbs) targeting EGFR (e.g., cetuximab and panitumumab) have shown remarkable efficacy in treating cases of mCRC in which the KRAS status is wild-type [3,4]. The American Society for Clinical Oncology (ASCO) and National Comprehensive Cancer Network (NCCN) guidelines both recommended that all mCRC tumors should be tested for KRAS mutations before beginning anti-EGFR mAb therapy [5,6]. The most frequent KRAS alterations associated with mCRC are detected in codons 12 and 13, and six codon 12 mutations account for more than 80% of the mCRC-associated KRAS mutations [7]. Thus, it is imperative to detect KRAS codon 12 mutations in a correct and timely fashion. However, clinical tumor samples are typically composed of both wild-type and mutant DNA, with the proportion of wild-type DNA often vastly exceeding that of the mutant. The PCR ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Zhao), [email protected] (W. Chen). 1 These authors contributed equally to this article.
sequencing method, which is generally considered a gold standard for clinical diagnosis, is reliable only when the mutant-to-wild ratio reaches 10%–20% [8,9]. Thus, more optimal KRAS mutation-testing methods are urgently needed to improve clinical diagnosis. To detect low-level mutations in tumors (e.g., 10−3 to 10−6 mutant to wild-type DNA), a method must have both high selectivity and the ability to enrich minority alleles. Over the past two decades, most of the research on enhancing the detection of minority (mutant) alleles in clinical samples has focused on improving the selectivity of PCRbased technologies and/or enriching the mutant sequences. A number of approaches have been developed, including amplification refractory mutation system (ARMS) [10,11], coamplification at lower denaturation temperature PCR (COLD-PCR) [12–14], locked nucleic acid/peptide nucleic acid clamp PCR (LNA/PNA-PCR) [15,16], mutant-enriched PCR [17,18], one-step enzyme-enriched ARMS PCR [19] and restriction endonuclease-mediated real-time digestion-PCR (RTD-PCR) [19]. Most of these assays were reported to show moderate to high selectivity. However, we do not yet have a universally accepted approach that can be used as a routine diagnostic tool, because it is difficult to achieve high selectivity and enrichment while maintaining accuracy, convenience, and low cost.
http://dx.doi.org/10.1016/j.clinbiochem.2014.08.015 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Xie F, et al, Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.015
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F. Xie et al. / Clinical Biochemistry xxx (2014) xxx–xxx
Here, we developed a one-step assay called real-time digestion-andblock TaqMan probe PCR (RTDB-PCR) for the detection of low-level KRAS codon 12 mutations within large excesses of wild-type KRAS sequences. The highlight of this technique lies in the use of PspGI, a thermostable endonuclease, and a MGB blocker. The wild-type DNA could be digested by PspGI under 75 °C, which made amplification and restriction digestion happen in one reaction. With the help of the MGB blocker, which completely matched the wild-type template, the specificity was further improved. We evaluated the feasibility of using RTDB-PCR on clinical tumor samples and compared it to the use of PCR sequencing, mutant-enriched PCR sequencing and mutantenriched PCR-MassArray. Materials and methods Ethics statement This research was approved by the Review Board of the Beijing Institute of Genomics, the Review Board of the Beijing Genomics Institute, and the Review Board of the National Institutes for Food and Drug Control of China. All participants gave written informed consent for use of their samples in research. Clinical samples A total of 100 tumor samples surgically resected from mCRC patients at the Peking Union Hospital (Beijing, China) were used in this study. Hematoxylin and eosin stained sections of formalin-fixed, paraffinembedded (FFPE) tissue from each sample were reviewed and the areas of highest tumor density (at least 50% tumor cells content) were identified and encircled. For each tumor, the FFPE block with the maximum number of tumor-rich areas was selected and sliced into two 5-μm-thick sections. A total of 60 blood samples from normal healthy controls were collected in EDTA-containing vacuum tubes at the Peking Union Hospital (Beijing, China); these were used to determinate the ΔCt cut-off value. Dilution panels and sensitivity evaluation
using a NanoDrop 8000 (Thermo, Germany) and ranged from 2 ng/μl to 100 ng/μl. Mutation testing methods RTDB-PCR A 141-bp product spanning codon 12 of KRAS exon 2 was amplified by primer F 5′-TAAACTTGTGGTAGTTGGACCT-3′ (forward, mismatched base is bolded and underlined) and primer R 5′-GGTCCTGCACCAGTAA TATGC-3′ (reverse). Thermostable PspGI was used to digest the amplified wild-type products. The MGB blocker (5′-TGGAGCTGGTGGCGT AGG-MGB-3′), which completely matched the wild-type template, was used to further improve the specificity. Signal generation was monitored in real-time using a TaqMan probe (5′-FAM-AGAGTGCCTT GACGATACAGCTAATTCA-BHQ1-3′). The reaction mixture consisted of 12.5 μl of 2 × Premix Ex Taq (TaKaRa), 5 U of PspGI (New England Biolabs, USA), 0.24 μM each of primers F and R, 0.4 μM of MGB blocker, 0.16 μM of TaqMan probe, 0.4 μl of 50 × ROX, 3 μl of templates, and ddH2O up to 25 μl. PCR was performed on an ABI 7300 real-time PCR instrument (Applied Biosystems, USA) according to the following protocol: 3 min of denaturation at 95 °C, followed by 40 cycles of 92 °C for 10 s, 55 °C for 35 s for primer annealing, extension and fluorescence data collection, and 75 °C for 60 s for PspGI cleavage. Each assay was performed in triplicate. The core of this technique is the use of the thermostable endonuclease, PspGI, and an MGB blocker. PspGI, which is purified from Pyrococcus sp. strain GI-H, is an extremely thermostable restriction endonuclease that has a half-life of 2 h at 95 °C. DNA containing the sequence 5′-CCWGG-3′ (W = A or T) is cleaved effectively by PspGI across an optimum temperature range of 75 °C to 85 °C [21]. In our strategy, a mismatched base was introduced into KRAS codon 11 (GCT NCCT) with primer F; once amplified, most of the wild-type KRAS (KRAS-wt) sequences (GCTGGTNCCTGGT) are cleaved at 75 °C for 1 min, enriching the mix for the mutant KRAS sequences (GCTGGT NCCTHHT H ≠ G). A blocker with an MGB label at the 3′ terminus was used to further improve the specificity; it was completely complementary to KRAS-wt and inhibited the amplification of wild-type (but not mutant) sequences. The principle of this design is depicted in Fig. 1.
DNA extraction
Determination of the ΔCt cut-off value To determine the specificity of our RTDB-PCR technique, we performed RTDB-PCR and control assays using 60 human genomic DNA samples from normal healthy controls with concentrations ranging from 2 ng/μl to 100 ng/μl. Reactions lacking PspGI and the MGB blocker were defined as control assays. All samples were assessed in three technical replicates. The ΔCt cut-off value was calculated by determining the average ΔCt between mutant-detecting and control assays and then subtracting a value of 3 times the standard deviation. This was done to ensure that there was a significant difference between true amplification of a mutant allele and non-specific, off-target amplification (according to the protocol provided with the ABI TaqMan® Mutation Detection Assay). The utilized formula was as follows: ΔCt cut-off = Average ΔCt-3 × the standard deviation and ΔCt = Ct (mutant assay) − Ct (control assay). If the ΔCt was below the ΔCt cut-off value, the sample was classified as positive. If the ΔCt was greater than the ΔCt cut-off value, the sample was classified as negative or beyond the limit of detection.
Tumor DNA was extracted from two sections of each sample using a QIAamp DNA FFPE Tissue kit (Qiagen, Germany) according to the manufacturer's instructions. DNA was eluted from the columns with 100 μl of water containing 20 μg/ml RNase A. After incubation at 37 °C for 15 min, the DNA was immediately stored at −20 °C. Normal genomic DNA was extracted from 200 μl of each blood sample using a TIANamp Blood DNA kit (Tiangen, China) according to the manufacturer's instructions. The concentrations of DNA were measured
PCR sequencing. PCR was performed in a 30-μl mixture containing 15 μl of 2× GC buffer (TaKaRa, Japan), 0.25 μΜ each of primers F and R, 1.5 U of rTaq, 4.8 μl of dNTP mixture (2.5 mM), 3 μl of template, and ddH2O up to 30 μl. PCR was performed on a 2720 PCR cycler (Applied Biosystems, USA) according to the following protocol: 3 min of denaturation at 95 °C, followed by 40 cycles of 92 °C for 10 s, 55 °C for 35 s and 72 °C for 60 s. The products were further sequenced using an ABI PRISM 3730xl system (Applied Biosystems, USA).
Recombinants containing the six most common mCRC-related KRAS codon 12 mutations and wild-type KRAS codon 12 were constructed using overlap extension PCR [20] and cloned into the pMD19-T plasmid (TaKaRa, Japan). Recombinants were extracted using TIANprep Mini Plasmid kits (Tiangen, China), and their sequences were confirmed by Sanger sequencing. All primers used in recombinant construction are listed in Supplementary Table 1. The concentration of recombinants was measured using a NanoDrop 8000 (Thermo, Germany). The mutant plasmid DNA was serially mixed with 15,000 copies/μl of wild-type plasmid DNA in the following percentages: 100%, 10%, 1%, 0.1%, 0.01%, and 0%, namely, mutant DNA content was 15,000 copies/μl, 1500 copies/μl, 150 copies/μl, 15 copies/μl of 1.5 copies/μl respectively. It was also serially diluted with 200 ng/μl human genomic DNA (SigmaAldrich, Cat. HRC1; 60,000 copies/μl) in the following percentages: 100%, 10%, 1%, 0.1%, 0.01%, and 0%. These two panels were used for sensitivity determination.
Please cite this article as: Xie F, et al, Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.015
F. Xie et al. / Clinical Biochemistry xxx (2014) xxx–xxx
5’ 3’
CCT CGANNA
5’
B
A Artificial introduction of restriction site 5’ 3’
CCTGGT
No introduction 3’ 5’
CCTGGT
No amplification
5’ 3’
5’ 3’
CCTGAT
3’ G12D 5’
5’ 3’
CCTGCT
3’ G12A 5’
5’ 3’
CCTGTT
3’ 5’ G12V
PO4 5’ 3’ 5’ 5’ 3’
CCTAGT
3’ G12S 5’
CCTCGT
3’ 5’ G12R
5’ 3’
CCTTGT
3’ 5’ G12C
3’ 5’
GCTGGT
No cleavage But blocked
Once amplified PspGI cleavage 5’ 3’
3
3’ 5’
MGB 3’
GCTGGT CGACCA
No amplification
All six mutants amplified with no difference Fig. 1. PspGI and MGB blocker-mediated KRAS mutation assay. (A) A mutagenic primer introduces a variation (denoted in red) that creates a recognition site for PspGI in the wild-type codon 12 target sequence, thereby suppressing the exponential amplification of the modified wild-type alleles. Meanwhile, the amplification of unmodified wild-type alleles is suppressed by a fully complementary MGB blocker. (B) The mutant alleles escape digestion and are exponentially amplified. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Mutant-enriched PCR sequencing. The reaction mixture was the same as described for RTDB-PCR, except that it lacked the MGB blocker and Taqman probe. The initial PCR conditions were as described for RTDBPCR. 5 μl PCR products were taken out for next Mutant-enriched PCRMassArray assay (Sequenom, USA) and the remaining 20 μl products were further sequenced using an ABI PRISM 3730xl system (Applied Biosystems, USA).
Mutant-enriched PCR-MassArray. For our mutant-enriched PCRMassArray assays, two separate well were used to assess the genotype of KRAS codon 12. Primers UEP1 5′-ACTCTTGCCTACGCCAC-3′ and UEP2 5′-CACTCTTGCCTACGCCA-3′ were designed as single-base extension primers for c.34G and c.35G, respectively. Mutant-enriched PCR products (5 μl; described above) were used as the template in order to minimize error between reactions. The unincorporated dNTPs were dephosphorylated by incubation at 37 °C for 40 min with 0.3 U of shrimp alkaline phosphatase (SAP), 0.17 μl of SAP buffer and 1.53 μl of ddH2O. Then, extension reactions were performed in a 9-μl system, as described in the Application Guide (Sequenom, USA). SAP and extension were run on a Veriti 384-well thermal cycler (Applied Biosystems, USA). Three replicate experiments were performed for each sample.
Statistical analysis To evaluate the performance of RTDB-PCR, the kappa values between RTDB-PCR and the other three methods were analyzed. Agreement between the pairs of tests was assessed using Kappa values, with values in the range of 0.00 to 0.20 indicating poor agreement, 0.21 to 0.40 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.80 good agreement, and 0.81 to 1.00 excellent agreement. All statistical calculations were performed with the Statistical Package for the Social Sciences (SPSS) software (version 16.0, SPSS Inc., Brussels, Belgium).
Results Patient and characteristics Of the tumors, 75% (75/100) were located in the colon, and the rest were located in the rectum. The average age of the overall population was 62 ± 12 (range, 28 to 89 years). The grade of differentiation of all 60 samples was documented. Further details on these patients are given in Table 1. Determination of the ΔCt cut-off value To calculate the ΔCt cut-off value for our RTDB-PCR, we tested 60 blood samples collected from healthy people at the Peking Union Hospital. All of the amplifications of these control assays were successful and their Ct values range from 20.18 to 27.86. As to mutant assays, wild-type DNA was digested completely by PspGI, so no detection signal
Table 1 Clinical characteristics of the patients.
Patient characteristics (+)a Sex Male (+) Female (+) Median age (range)b Grade of differentiation Poorly (+) Moderately (+) Well (+) Lymph node metastasis Yes (+) No (+)
Colon cancer
Rectal cancer
Total
75(34)
25(11)
100(45)
45(20) 30(14) 61 ± 13(28–89)
13(5) 12(6) 63 ± 10(44–79)
58(25) 42(20) 62 ± 12(28–89)
10(6) 23(9) 42(19)
2(1) 10(3) 13(7)
12(7) 33(12) 55(26)
33(17) 42(17)
13(6) 12(5)
46(23) 54(22)
Note: a Represents the number of RTDB-PCR positive samples. b Represents the range of patient's age.
Please cite this article as: Xie F, et al, Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.015
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F. Xie et al. / Clinical Biochemistry xxx (2014) xxx–xxx
was collected. Then we defined 40 as the Ct of mutant assay and calculated the ΔCt cut-off value as following: ΔCt cut‐off ¼ Average ΔCt−3 the standard deviation ¼ 16:5−3 1:63 ¼ 11:6:
as calculated by the peak area ratio of mutant to wild-type in the mutant-enriched PCR-MassArray assay (Supplementary Table 2). A sensitivity comparison of these four methods against the 15,000 copies/μl of wild-type plasmid background was depicted in Fig. 2.
Mutation testing of clinical specimens Sensitivity comparison Only RTDB-PCR was sensitive to 0.01% mutant DNA in both backgrounds (15,000 copies/μl of wild type plasmid DNA or 200 ng/μl human genomic DNA). The sensitivity of the mutant-enriched PCRMassArray reached 0.01% in the 200 ng/μl DNA panel, but was only 0.1% in the 15,000 copies/μl of DNA panel. The sensitivity of the mutant-enriched PCR sequencing was 0.1% in both panels, while that of PCR sequencing was only 10% in both panels. After PspGI digestion, the fold of mutant enrichment was as high as 2975 (29.75% to 0.01%),
Comparison between RTDB-PCR and PCR sequencing All 100 of the mCRC FFPE samples received from Peking Union Hospital were successfully tested by RTDB-PCR, and KRAS codon 12 mutations were found in 45 samples. In contrast, only 26 mutations were identified by PCR sequencing. The positive agreement, negative agreement and overall agreement between RTDB-PCR and PCR sequencing were 100%, 74.4% and 81%, respectively, and the kappa value between the two methods was 0.601 (Table 2). We did a simple classification of 45 RTDB-PCR positive samples and found that 34 were
PCR sequencing
RTDB-PCR 100%
IC
IC 0.1%
1%
10%
0.01%
IC
IC
100%
10%
1%
0.1%
0.01%
0%
IC 0%
IC
A
B
Mutant-enriched PCR-MassArray
Mutant-enriched PCR sequencing 100%
10%
1%
W
M
100%
0.1%
0.01%
0%
W
C
M
W
10%
M
0.1%
failure
W
W
M
1%
M
0.01%
M
W
0%
failure
D
Fig. 2. Sensitivity evaluation of RTDB-PCR, PCR sequencing, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray. (A) to (D) respectively described the KRAS G12D sensitivity detection plot of RTDB-PCR, PCR sequencing, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray in a background of 15,000 copies/μl of wild-type plasmids. The proportion of mutant to wild plasmid ranged from 100% to 0%.(A) IC indicated the control assay without PspGI or the MGB blocker. The curves indicated by a red arrow represent the amplification plot of RTDB-PCR detection and 0.01% mutants could be detected by RTDB-PCR. (B) and (C) showed the results of PCR sequencing and mutant-enriched PCR sequencing. Mutant peak was indicated by a red arrow. The sensitivity of the mutant-enriched PCR sequencing was 0.1%, while that of PCR sequencing was only 10%. (D) The extension production peak of mutant-type and wild-type was marked with M and W. The sensitivity of the mutant-enriched PCR-MassArray reached 0.1% in the 15,000 copies/μl of DNA background.
Please cite this article as: Xie F, et al, Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.015
F. Xie et al. / Clinical Biochemistry xxx (2014) xxx–xxx Table 2 Comparison of RTDB-PCR and PCR sequencing. N = 100
PCR sequencing
RTDB-PCR + − Total
+
−
Total
26 0 26
19 55 74
45 55 100
colon cancer and 11 were rectal cancer. More details of the classification of RTDB-PCR positive samples were shown in Table 1. Comparison of RTDB-PCR, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray RTDB-PCR, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, which all use thermostable PspGI to improve their selectivity, successfully identified 45, 44 and 44 samples, respectively. There were one sample tested positive to RTDB-PCR but it has a ΔCt value with 10.68. It suggested that it has very low mutations. The positive agreement, negative agreement and overall agreement between RTDB-PCR and either of the other two methods were 100%, 98.2%, 99%, respectively, and the kappa value in each case was 0.980 (Table 3). 44 of 45 mutant specimens detected by RTDB-PCR were also genotyped based on the sequencing data obtained from the mutantenriched PCR MassArray and mutant-enriched PCR sequencing methods. Among these 44 cases, there were 36 single-base mutations and 8 complex mutations, as follows: 14 G12D (GGT NGAT), 11 G12A (GGT N GCT), 7 G12V (GGT N GTT), 3 G12C (GGT NTGT), 1 G12S (GGT AGT), 4 G12D/G12A (GGTNGAT/GCT), 1 G12V/G12A (GGTNGTT/GCT), 1 G12C/G12A (GGTNTGT/GCT), 1 G12D/G12 C(GGTN GAT/TGT), and 1 G12D/G12A/G12V (GGTNGAT/GCT/GTT). Discussion In the wake of the development of molecular targeted therapy, it has become increasingly important to detect biomarkers quickly, sensitively and accurately [22]. For mutation detection, Sanger sequencing has been widely utilized and still serves as a gold standard in most clinical laboratories, but lower sensitivity limited its utility [23,24]. It is very difficult to obtain homogeneous tumor samples in the clinical setting. Even if we could obtain homogeneous tumor samples, the somatic mutations were also heterogeneous [25]. Mutation content was often so little that the limit of detection of PCR sequencing couldn't reach. Novel diagnostic techniques that purportedly have high selectivity are constantly springing up. However, we do not yet have a uniform standard diagnostic method for detecting mutations, and most of the “highly sensitive” methods that have been reported are laborious, time-consuming, expensive, and/or limited by issues with low template concentrations [26]. In the present study, our RTDB-PCR had higher sensitivities and was able to detect as few as 0.01% mutants in a background of 200 ng wildtype DNA. It is a huge improvement over the sensitivity of PCR sequencing which could detect as few as 10% mutants. The agreement between RTDB-PCR and PCR sequencing for 100 mCRC sample detection was
Table 3 Comparison of RTDB-PCR and the other two enriched methods. N = 100
RTDB-PCR + − Total
Mutant-enriched PCR sequencing
Mutant-enriched PCR-MassArray
+
−
+
−
44 0 43
1 55 56
44 0 43
1 55 56
Total
45 55 100
5
poor (the kappa value was only 0.601), mainly because of the low sensitivity of PCR sequencing. Compared with mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, RTDB-PCR has an equal or slight higher sensitivity. Mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray both detect 44 positive samples, while RTDB-PCR tested one more positive sample (control Ct = 26.16, detection Ct = 36.84, ΔCt = 10.68). In sensitivity assays, we found mutant-enriched PCR sequencing and mutant-enriched PCRMassArray with only 0.1% sensitivity in the 15,000 copies/μl of DNA panel. The ΔCt value of this sample (contains about 5000 copies/μl of total DNA) was greater than the ΔCt value (ΔCt values approximately equal 10) of 0.1% sensitivity, leading to a negative (failed testing) result. As a high-throughput and cost effective method, MassArray iPLEX assay can analyze up to 384 samples with one multiplex assay or up to 384 different multiplexed assays with one sample (see MassARRAY iPLEX® Gold SNP Genotyping instruction). However, mutant-enriched PCR-MassArray requires a separate pre-PCR because of PspGI digestion, which produces a complex operation. Besides, PCR-MassArray needs a relatively high DNA concentration for spotting, which also limits its application especially in low level mutation testing. Mutant-enriched PCR-MassArray and mutant-enriched PCR sequencing could discriminate the specific genotypes of KRAS mutation. After a comprehensive analysis, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray are less suitable for clinical testing because: 1) no evidence showed that the specific genotypes of codons 12 and 13 have different contributions to anti-EGFR mAb therapy; 2) it only include a closed-tube detection and have less contamination risks than mutantenriched PCR sequencing and mutant-enriched PCR-MassArray; 3) it would only take about 2 h whereas mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray would take more than 24 h to finish the tests; and 4) mutant-enriched PCR sequencing and mutantenriched PCR-MassArray both need expensive instruments which cost more than 300,000 USD. Therefore, our novel RTDB-PCR method seems to be a powerful new tool for mutation screening early during disease progression, and thus could have important therapeutic implications. Conflict of interest The authors declare no conflict of interest. Acknowledgments We are grateful to the Peking Union Hospital for providing tissue samples. This project was supported by the Major State Basic Research Development Program of China (grant No. 2010CB529101, 973 Program) and the Chinese 863 Program (2012AA02A201). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.clinbiochem.2014.08.015. References [1] Maughan TS, Adams RA, Smith CG, Meade AM, Seymour MT, Wilson RH, et al. Addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial. Lancet 2011;377:2103–14. [2] Tveit KM, Guren T, Glimelius B, Pfeiffer P, Sorbye H, Pyrhonen S, et al. Phase III trial of cetuximab with continuous or intermittent fluorouracil, leucovorin, and oxaliplatin (Nordic FLOX) versus FLOX alone in first-line treatment of metastatic colorectal cancer: the NORDIC-VII study. J Clin Oncol 2012;30:1755–62. [3] Lievre A, Bachet JB, Le Corre D, Boige V, Landi B, Emile JF, et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res 2006; 66:3992–5. [4] Van Cutsem E, Kohne CH, Hitre E, Zaluski J, Chang Chien CR, Makhson A, et al. Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med 2009;360:1408–17.
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Please cite this article as: Xie F, et al, Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.015
