Lung cancer is the most common cause of cancer related death in the world, particularly in major cities in China. The majority of these deaths are due to non-small cell lung cancer (NSCLC) which is the most common histologic type.1 Routine platinum based chemotherapy has been used to treat patients with advanced NSCLC, but prognosis is particularly poor for these patients.2,3 Therefore effective low-toxicity treatments for patients with advanced NSCLC are needed.
It has been proved that expression of the epidermal growth factor receptor (EGFR) is frequently found in NSCLC and plays a central role in development of NSCLC. Tyrosine kinase inhibitors (TKIs) that target EGFR have demonstrated effectiveness in treatment of NSCLC patients.4,5 EGFR TKI efficacy has been associated with non-smokers, women, Asian ethnicity and adenocarcinoma histology. 6 Furthermore, a correlation between mutations in the EGFR gene's kinase domain and sensitivity to EGFR TKIs has been reported and subsequently validated in several clinical trials.7-9 Most EGFR mutations are observed in exons 19 and 21. This important breakthrough indicated that EGFR mutations can be used as a biomarker for screening patients, particularly Chinese patients with high EGFR mutation frequencies, for treatment with EGFR TKIs.
EGFR mutations can be easily detected in tumor tissues. However, it may be difficult to obtain tumor tissues from patients with advanced NSCLC. Plasma samples of patients with NSCLC, especially with advanced NSCLC or progressive disease, often contain circulating DNA derived from tumor tissues,10,11 so plasma samples have been designated as surrogate tumor tissues for detecting genetic alterations.12-14 Several studies have shown the consistency of EGFR mutations between tissue and serum DNA.15
Because of the low concentration of tumor DNA in circulating blood, detecting EGFR mutations in plasma DNA samples from patients with advanced NSCLC is challenging. In order to find a sensitive and specific method for detection of EGFR gene mutations in circulating blood, we compared three methods for detecting plasma EGFR mutations, including direct DNA sequencing, denaturing high-performance liquid chromatography (DHPLC) and Scorpions Amplification Refractory Mutation System (Scorpions ARMS).
Patients treated at the Peking Union Medical College Hospital from August 2008 to May 2009 were enrolled in this study. Seventy-three stage IIIB to IV NSCLC patients with confirmed adenocarcinoma histology were analyzed for EGFR mutations in exons 19 (deletion mutation) and 21 (L858R mutation). Laboratory data were obtained and recorded independently by investigators blinded to clinical data until analyses were undertaken. The study was reviewed and approved by the Institutional Ethic Committee at Peking Union Medical College Hospital. All the patients signed informed consent form to participate in this study and gave permission for the use of their plasma.
Specimen collection, DNA extraction and nest polymerase chain reaction (PCR)
Plasma samples were collected from patients and the samples were digested with proteinase K and genomic DNA was extracted using the phenolchloroform extraction method. DNA was precipitated with ethanol and then precipitated DNA was eluted in 50 of sterile, double-distilled water. The DNA was stored at -20°C and used for subsequent analysis.
To ensure the specificity of PCR, nested PCR was performed to amplify exons 19 and 21 of EGFR. The first round PCR reaction mixture containing 1 μl of 10× buffer, 0.2 μl dNTPs (10 mmol), 0.2 μl (5 mmol) forward and reverse outer primers respectively, 0.2 U of Taq polymerase (Promega, Madison City, USA), 20 ng DNA sample, and ddH2O was added to a final volume of 10 μl. The second round PCR reaction mixture containing 3 μl 10× buffer, 0.6 μl dNTPs (10 mmol), 0.6 μl (5 mmol) forward and reverse inner primers respectively, 0.2 U of Taq polymerase (Promega), 2 μl DNA products amplified by the outer primers, and ddH2O was added to a final volume of 30 μl. The sequences of inner and outer primers were designated according to Bai et al (Table 1).15
The conditions for amplification with outer primer sets were at 95°C for 5 minutes, followed by 5 cycles at 94°C for 30 seconds, at 56°C for 30 seconds, and at 72°C for 30 seconds. This was followed by 20 cycles at 94°C for 30 seconds, at 61°C for 30 seconds, and at 72°C for 30 seconds, and a single extension cycle at 72°C for 10 minutes. The conditions for amplification with inner primer sets were at 95°C for 5 minutes, followed by 35 cycles at 94°C for 30 seconds, at 60°C for 30 seconds, and at 72°C for 30 seconds, and a single extension cycle at 72°C for 10 minutes. PCR products were resolved on a 2.5% agarose gels stained with ethidium bromide and observed under ultra-violet (UV) illumination.
Nested PCR products for EGFR exons 19 and 21 were gel-purified using a PCR fragment purification kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. The PCR products were then sequenced directly with the BigDye terminator cycle sequencing kit (ABI, Foster, USA) and were analyzed on an ABI automated sequencer. Sequencing reactions were performed on both DNA strands, using the inner primers of EGFR exons 19 and 21.
DHPLC was performed using the Transgenomic Wave Nucleic Acid Fragment Analysis System with a DNASep column (Transgenomic, Omaha, NE, USA). The mobile phases were comprised of 0.05% acetonitrile in 0.1 mol/L triethylammonium acetate (TEAA; eluent A) and 25% acetonitrile in 0.1 mol/L TEAA (eluent B). The PCR products of exons 21 were denatured at 95°C for 5 minutes and were cooled to 35°C at a rate of 1°C per minute to allow formation of heterozygote DNA. The product of exon 19 did not need to be denatured. The flow rate was 0.9 ml/min, and the ultraviolet detector was set at 260 nm. The heterozygous profiles were identified by visual inspection of the chromatograms on the basis of the appearance of additional, earlier eluting peaks. Corresponding homozygous profiles showed only one peak.
The EGFR Scorpions kit was used to detect deletion mutations of EGFR exon 19 and L858R mutations of EGFR exon 21 in real-time PCR reactions. All reactions proceeded in 25 μl volumes using 1 μl template DNA, 7.5 μl reaction buffer mix, 0.6 ml primer mix, and 0.1 ml Taq polymerase. Real-time PCR was performed using Bio-Rad iQ5 real-time PCR system (Bio-Rad Laboratory, USA) under the following conditions: initial denaturation at 95°C for 10 minutes, 50 cycles of 95°C for 30 seconds, and 61°C for 60 seconds with fluorescence reading (set to FAM, which allows optical excitation at 480 nm and measurement at 520 nm) at the end of each cycle. Data were analyzed using Bio-Rad iQTM5 software (version 2.0). The cycle threshold (Ct) was defined as the cycle at the highest peak of the second derivative curve that represented the point of maximum curvature of the growth curve. Both Ct and maximum fluorescence were used for interpretation of the results. Positive results were defined as Ct ≤45 and maximum fluorescence intensity ≥30. When only the curve that indicated the wild-type increased, the sample was considered the wild-type EGFR. When both wild- and mutation-type curves increased, the sample was considered the mutant-type EGFR.
Data were analyzed using SPSS statistical software, version 13.0. The chi-square or Fisher's exact tests were used to compare the sensitivities of three methods. Kappa test was used to compare the consistence of different methods. The time to event variables (ie, overall survival (OS) and progression-free survival (PFS)) were calculated by Kaplan-Meier estimation. Probability value was obtained from two-sided tests, with statistical significance defined as P <0.05.
During this study, all 73 patients with advanced adenocarcinoma received chemotherapy and 46 of them received second-line gefitinib treatment. The patients' clinical characteristics are listed in Table 2.
Direct gene sequencing analysis results
EGFR mutations were observed in 5 samples by direct DNA sequencing, 2 deletion mutations in exon 19 and 3 L858R mutations in exon 21 (Figure 1).
DHPLC analysis results
A total of 22 samples (30.1%) were detected with EGFR mutations by DHPLC. On DHPLC analysis, the EGFR exon 19 deletion exhibited two peaks. The eluting peak of the wild-type sequence arrived at 5 minutes and 1 second, whereas the peak of the deletion mutant arrived at 3 minutes and 30 seconds (Figure 2A). The EGFR exon 21 mutation exhibited an abnormal overshoot at the peak of the wild-type (heterozygous peaks), whereas the wild-type alleles showed a sharp peak pattern (Figure 2B). Of the 73 patients, EGFR mutations were found by DHPLC in 13 samples in exon 19 and in 9 samples in exon 21.
Scorpions ARMS analysis results
Scorpions ARMS analysis of EGFR mutation are shown in Figure 3. The wild type showed one increased curve, and mutant type showed three increased curves. Using the EGFR Scorpions kit, 15 deletion mutations in exon 19 and 13 L858R mutations in exon 21 of EGFR were detected.
Comparison between gene sequencing, DHPLC and Scorpions ARMS
Because there were only mutations in the EGFR in five patients detected by the direct gene sequencing method, gene sequencing was clearly less sensitive than the other two methods tested in our study. In 73 patients, EGFR mutations were detected in 22 samples (30.1%) by DHPLC, and in 28 samples (38.4%) by Scorpions ARMS. Although Scorpions ARMS seems more sensitive than DHPLC, there was no significant difference between them (χ2=1.095, P=0.295). Among the test results from 73 patients, there was a 90.4% concordance between the DHPLC and Scorpions ARMS methods (66/73, k=0.79, P=0.07). Seven patients were detected with a discordant mutation status by DHPLC and Scorpions ARMS. Six EGFR mutation-positive samples detected by Scorpions ARMS were found mutation-negative by DHPLC, and one EGFR mutation-positive sample detected by DHPLC was found mutation-negative by Scorpions ARMS.
Correlation between EGFR mutation and survival
According to the results of the Scorpions ARMS assay, we analyzed the potential implication of EGFR mutation status in predicting clinical outcome in the patients with adenocarcinoma who received EGFR-TKI. The follow-up time was calculated from the start of EGFR-TKI treatment. The median follow-up time of these 46 patients was 25.0 months (from 1.0 to 120.0 months). There were 18 patients with mutations and 28 patients without mutations as determined by Scorpions ARMS. The 18 patients with mutations had a significantly longer PFS time (median PFS was 21.0 months), than the 28 patients without mutations (median PFS was 7.0 months) (P=0.022) (Figure 4A). Similar results were obtained when we used the DHPLC method (Figure 4B). An association between EGFR mutation status and OS was not observed because the follow-up time was not long enough to determine OS.
In this study we demonstrated the feasibility of detecting EGFR mutations in circulating blood samples from NSCLC patients. In order to find an accurate and economic method to detect EGFR mutations in circulating blood samples, our study compared three methods: gene sequencing, DHPLC and Scorpions ARMS. Direct gene sequencing showed the lowest sensitivity among the three methods. Gene sequencing had been regarded as a “gold” standard for gene mutation analysis, however, it was not sensitive enough to detect gene mutations in DNA found in plasma or serum samples of NSCLC patients because of the low percentage of tumor DNA in the circulating blood.
Recently, new methods with high sensitivity, such as DHPLC, Scorpion ARMS, and mutant-enriched PCR assay, have been used in the field of gene mutation analysis. DHPLC and Scorpions ARMS were extensively used to detect mutations in both tumor tissues and plasma or serum samples. Both methods showed higher sensitivity when compared to gene sequencing, which is consistent with previous reports.16-18 In our study, the frequency of EGFR mutations (deletion mutation and L858R mutation) detected by DHPLC and Scorpions ARMS was 30.1% and 38.4%, respectively. Scorpions ARMS seems more sensitive than DHPLC in this study. Among the 73 patients, there was a 90.4% concordance between DHPLC and Scorpions ARMS. Our results were consistent with other studies,17,18 and proved the utility of both technologies in clinical practice. Our findings of a correlation between EGFR mutations and tumor response to TKI treatment was also consistent with previously reported data.19,20 In our patient population, the difference in OS was not obtained because OS time was not reached. We will continue to follow this group of patients. Patients with EGFR mutations detected by either DHPLC or Scorpions ARMS had significantly longer PFS time after EGFR-TKI treatment, suggesting that these patients might have benefited from the treatment. However, it should be noted that our study was not specifically designed to test EGFR-TKI treatment and that many patients also received other chemotherapeutic agents, which makes data interpretation difficult.
A number of factors will impact the use of genetic tests in routine clinical practice, including the technical complexity, sensitivity of the technique, and cost of the tests. These factors are particularly important for countries such as China, where the patient population is large and health care resources are limited. In the present study, among the three methods for detecting EGFR mutations in plasma DNA samples in patients with advanced lung adenocarcinoma, direct gene sequencing had the lowest sensitivity, while Scorpion ARMS showed the highest mutation detecting capability. Although DHPLC is slightly less sensitive than Scorpion ARMS, the DHPLC method utilized in this study is technically easier and less expensive. It has been evaluated for EGFR mutation analysis by other investigators.15,17 Our results support the utility of this technology in routine clinical practice.
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