The Journal of International Medical Research 2011; 39:

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1 The Journal of International Medical Research 2011; 39: Serum Detection of Epidermal Growth Factor Receptor Gene Mutations Using Mutant-enriched Sequencing in Chinese Patients with Advanced Non-small Cell Lung Cancer B JIANG 1,2, F LIU 2, L YANG 3, W ZHANG 2, H YUAN 2, J WANG 2 AND G HUANG 1 1 Department of Nuclear Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2 Department of Medical Oncology, No. 3 People s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 3 Department of Medical Oncology, Changhai Hospital, Shanghai Second Military Medical University, Shanghai, China Epidermal growth factor receptor gene (EGFR) mutations are among the best predictive markers of the efficacy of EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment. Mutations in the EGFR gene confer sensitivity to EGFR-TKIs in patients with advanced non-small cell lung cancer (NSCLC). This study determined the concordance rate of EGFR mutations in serum samples and tumour tissue from Chinese patients with advanced NSCLC and compared two detection methods: mutant-enriched polymerase chain reaction-based DNA sequencing and nonenriched sequencing. The EGFR mutation status in serum was consistent with that in paired tumour samples, with a concordance rate of 93.1% for mutantenriched sequencing. In serum samples, mutant-enriched sequencing demonstrated sensitivity and specificity of 77.8% and 100%, respectively, and was more sensitive than the non-enriched assay. Mutant-enriched sequencing in serum may provide a non-invasive and sensitive method for detecting EGFR mutation status in patients with unresectable NSCLC. KEY WORDS: NON-SMALL CELL LUNG CANCER; EPIDERMAL GROWTH FACTOR RECEPTOR; MUTATION; POLYMERASE CHAIN REACTION; MUTANT-ENRICHED SEQUENCING; SERUM; TISSUE Introduction Lung cancer continues to be the leading cause of cancer deaths worldwide, and nonsmall cell lung cancer (NSCLC) accounts for about 80% of cases. 1,2 Inhibitors of epidermal growth factor receptor tyrosine kinase (EGFR-TKIs) show clinical efficacy, compared with the best supportive care or standard chemotherapy, when given as second- or third-line therapy for advanced or metastatic NSCLC. 3 8 Because increasing evidence indicates that activation of mutations in the EGFR gene confers sensitivity to the EGFR-TKIs gefitinib and 1392

2 erlotinib in patients with advanced NSCLC, EGFR mutations are considered to be among the best predictive markers of the efficacy of EGFR-TKI treatment The two most common NSCLC-associated EGFR mutations are the 15 base-pair inframe deletion in exon 19 (E746_A750del) and the point mutation replacing leucine with arginine at codon 858 in exon 21 (L858R) These two mutations account for approximately 90% of all EGFR mutations and can explain the dramatic response to EGFR-TKIs. 17 It has been suggested that EGFR mutation status should be determined before the initial treatment of pulmonary adenocarcinoma whenever possible. 18,19 Such diagnostic analysis of genetic mutations appears particularly important for East Asians because their EGFR mutation frequency is higher than that in Caucasians (30 40% versus 10%). 20 However, obtaining tumour tissues for mutation analysis is challenging. Sufficient tissue biopsy is not obtainable in a considerable proportion of patients and most patients who require gefitinib therapy are already at an advanced stage of disease at the time of diagnosis and, as a result, are not operated on. It is, therefore, important to use sensitive methods to look for EGFR mutations in patient samples that are more readily accessible. Because blood samples of cancer patients often contain DNA derived from tumour tissues and significantly higher DNA levels are found in the serum of patients with metastatic disease, serum samples have been used as surrogate tumour tissues for detecting genetic alterations. 21 It has been reported that it is feasible to use serum DNA to detect EGFR mutation status and evaluate its potential as a predictor of response to EGFR-TKIs. 21,22 The detection of EGFR mutations in serum DNA may provide a non-invasive and repeatable source of genotypic information that could influence treatment and prognosis, especially in patients with NSCLC treated with EGFR- TKIs. In Chinese patients with advanced NSCLC, however, the use of serum DNA to detect EGFR mutations is still under development. Bai et al. 23 have reported that, by using denaturing high-performance liquid chromatography (dhplc), EGFR mutations can be detected reliably in plasma DNA of Chinese patients. However, this method is limited to screening for heterozygous variants and lacks the ability to identify deletions and duplications, which requires semi-quantitative polymerase chain reaction (PCR) and direct sequencing for verification. 24 In addition, although it has been supposed that amplification of DNA by mutant-enriched PCR is a more sensitive method in tumour and pleural fluid specimens, the feasibility of using mutant-enriched PCR to detect EGFR mutations in serum samples remains unclear. 25 The aims of this study were (i) to determine the concordance rate of EGFR mutations in serum samples and tumour tissue from Chinese patients with advanced NSCLC, and (ii) to compare two detection methods: mutant-enriched PCR-based DNA sequencing and non-enriched sequencing. Patients and methods PATIENTS Chinese patients with stage IIIB or IV NSCLC met the enrolment criteria and were entered into the study between May 2006 and September 2008 at three hospitals (Renji Hospital, Shanghai Jiao Tong University School of Medicine, No. 3 People s Hospital, Shanghai Jiao Tong University School of Medicine, and Changhai Hospital, Shanghai 1393

3 Second Military Medical University). None of the patients received chemotherapy or radiotherapy before recruitment. Histological diagnosis was made, using resected specimens, by the Pathology Department of No. 3 People s Hospital, Shanghai Jiao Tong University School of Medicine. Patient characteristics, comprising age, sex, smoking habit and histological tumour type, were recorded. Patients who had smoked < 100 cigarettes in their lifetime were categorized as never smokers, patients who had smoked > 100 cigarettes within 1 year of diagnosis were categorized as current smokers and patients who had smoked > 100 cigarettes in their lifetime but had stopped smoking 1 year before diagnosis were categorized as former smokers. Written informed consent was obtained from all patients prior to their inclusion. The study was approved by the ethics review committees of the institutional review boards of the three hospitals involved in the study. SAMPLES Samples of tumour tissue were obtained from the patients by computed tomographyguided transthoracic needle lung biopsy, electron tracheoscope-guided transbronchial lung biopsy or lymph node biopsy. All specimens were examined histologically to confirm the diagnosis of NSCLC. DNA was extracted from five serial 10 µm thick sections using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer s instructions. Matched blood samples (5 ml) were also collected from the patients before treatment. Serum was separated within 2 h after sample collection and stored at 80 C until analysis. DNA was isolated from serum using a QIAamp DNA Blood Kit (Qiagen) according to the manufacturer s instructions. MUTANT-ENRICHED SEQUENCING FOR EGFR MUTATIONS IN EXON 19 The mutant-enriched PCR assay is a two-step PCR procedure with intermittent restriction enzyme digestion selectively to eliminate wild-type alleles, thus enriching the mutated alleles. The primer sequences for PCR amplification were: forward primer 1 (ex19- F1), 5 -CCA GAA GGT GAG AAA GAT AAA ATT C-3 ; reverse primer 1 (ex19-r1), 5 -GTG GGC CTG AGG TTC AGA G-3 ; forward primer 2 (ex19-f2), 5 -ACT GTA AAA CGA CGG CCA GTG GTG AGA AAG ATA AAA TTC CCG TCG CTA T-3 ; reverse primer 2 (ex19- R2), 5 -ACC AGG AAA CAG CTA TGA CCC CCC ACA CAG CAA AGC AGA AAC TCA CAT-3. Each sample (10 ng of genomic DNA) was first amplified using the ex19-f1 and ex19-r1 primers in a PCR protocol consisting of one cycle of 95 C for 10 min and 30 cycles of 94 C for 30 s, 56 C for 60 s and 72 C for 40 s, followed by one cycle of 72 C for 10 min. After the first round of PCR, 2 µl of PCR products were digested with 10 U of MseI at 37 C for 4 h. MseI can recognize and cut the TTAA sequence in wild-type DNA but not in mutant DNA with exon 19 deletions, resulting in mutant-enriched DNA. Because the TTAA sequence was also present 26 bases upstream of codon 747, a forward mismatch primer was used to create a one-base mismatched (ATAA, T A) sequence to protect the primer from digestion. After MseI digestion, a second PCR was done using the ex19-f2 and ex19-r2 primers. The PCR conditions for the second PCR were one cycle of 95 C for 10 min and 35 cycles of 94 C for 30 s, 58 C for 30 s and 72 C for 50 s, followed by one cycle of 72 C for 10 min. The product of the second amplification was sequenced using the Applied Biosystems BigDye Terminator sequencing method with an ABI 3730XL DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA). 1394

4 MUTANT-ENRICHED SEQUENCING FOR EGFR MUTATIONS IN EXON 21 The mutant-enriched PCR assay for exon 21 was similar to the method described above for exon 19 except for the primer sequences and restriction enzyme. The primer sequences for PCR amplification were: forward primer 1 (ex21-f1), 5 -CCT GGC AGC CAG GAA CGT AC-3 ; reverse primer 1 (ex21-r1), 5 -GCC TGG TCC CTG GTG TCA GGA A-3 ; forward primer 2 (ex21-f2), 5 - ACT GTA AAA CGA CGG CCA GTA CAC CGC AGC ATG TCA AGA TCA C-3 ; reverse primer 2 (ex21-r2), 5 -ACC AGG AAA CAG CTA TGA CCG GTC CCT GGT GTC AGG AAA ATG-3. Genomic DNA was first amplified using the ex21-f1 and ex21-r1 primers. The first-step PCR protocol was one cycle of 95 C for 10 min and 35 cycles of 94 C for 30 s, 57 C for 40 s and 72 C for 50 s, followed by one cycle of 72 C for 10 min. After the first round of PCR, 2 µl of PCR products were digested with 10 U of MscI at 37 C for 4 h. MscI was used to digest the TGGCCA sequence in wild-type DNA. The mutant type (L858R) was not digested because of the T G substitution at the first base of TGGCCA, resulting in enrichment of the L858R mutant. After digestion, the second PCR was done using the ex21-f2 and ex21-r2 primers. The PCR conditions for the second PCR were one cycle of 95 C for 10 min and 40 cycles of 94 C for 30 s, 40 C for 40 s and 72 C for 50 s, followed by one cycle of 72 C for 10 min. The product of the second amplification was sequenced as described above. NON-ENRICHED SEQUENCING FOR EGFR MUTATIONS IN EXONS 19 AND 21 Samples were also analysed with a nonenriched PCR-based assay. The same sets of primers, PCR steps and cycles as those used for mutant-enriched sequencing were used for non-enriched sequencing for exons 19 and 21. The products of the first amplification were not digested by the restriction enzyme and the products of the second amplification were sequenced. These PCR-based and direct sequencing assays were regarded as the non-enriched assay. STATISTICAL ANALYSES Associations between the presence of EGFR mutations and patient characteristics, comprising sex, tumour histology and smoking habit, were analysed using Fisher s exact test. All statistical analyses were performed using SAS (version 8.0, SAS Institute, Cary, NC, USA). A two-tailed P- value < 0.05 was considered significant. Results PATIENTS A total of 58 Chinese patients with stage IIIB or IV NSCLC participated in the study and their clinical characteristics are shown in Table 1. Fifty-eight samples of tumour tissue were obtained from the patients using computed tomography-guided transthoracic TABLE 1: Clinical characteristics of the 58 patients with non-small cell lung cancer included in the present study Age, years Median 56 Range Sex, n (%) Male 40 (69.0) Female 18 (31.0) Smoking history, n (%) Never 22 (37.9) Former 6 (10.3) Current 30 (51.7) Histology, n (%) Adenocarcinoma 42 (72.4) Squamous cell carcinoma 14 (24.1) Large cell carcinoma 2 (3.4) 1395

5 needle lung biopsy (38 samples), electron tracheoscope-guided transbronchial lung biopsy (12 samples) or lymph node biopsy (eight samples). MUTANT-ENRICHED SEQUENCING FOR EGFR MUTATIONS IN EXONS 19 AND 21 Deletions in exon 19 and L858R mutations in exon 21 in EGFR were detected in 10 (17.2%) and eight (13.8%) of the 58 tumour samples, respectively. The same EGFR mutations were detected in both tumour and corresponding serum samples of 14 patients. EGFR mutations were not detected in either the tumour sample or the serum sample in 40 patients. The concordance rate was 93.1% (54 of 58 pairs). The sensitivity and specificity for mutantenriched sequencing in serum samples was 77.8% (14/18) and 100% (14/14), respectively. EGFR mutations were detected in tumour samples but not in corresponding serum samples in four patients (Table 2). ASSOCIATION BETWEEN EGFR MUTATION STATUS AND CLINICAL CHARACTERISTICS The EGFR mutations were significantly more frequent in tumour tissue samples from never smokers than in those from current/former smokers (54.5% [12/22] versus 16.7% [6/36] of cases, P = ) and in females than in males (66.7% [12/18] versus 15.0% [6/40], P < 0.001) (Table 3). Consistent with the results from tumour tissue samples, EGFR mutations were also more frequent in serum samples from the never-smokers than in those from current/former smokers (45.5% [10/22] versus 11.1% [4/36], P = ) and in females than in males (55.6% [10/18] versus 10.0% [4/40], P < 0.001). The EGFR mutations were found in all patients with adenocarcinoma included in this study. MUTANT-ENRICHED AND NON- ENRICHED SEQUENCING IN TUMOUR AND SERUM The EGFR mutation status in both tumour and serum samples was further examined using non-enriched sequencing and compared with the results obtained from mutant-enriched sequencing. In the tumour specimens, deletions in exon 19 and L858R mutations in exon 21 were detected in 18 of 58 samples using non-enriched sequencing, which was consistent with the results from the mutant-enriched assay. When using the non-enriched assay to analyse the serum samples, deletions in exon 19 and L858R mutations in exon 21 were detected in seven and four specimens, respectively. The concordance rate for the non-enriched assay was 87.9% (51/58 pairs). The sensitivity and specificity for non-enriched sequencing in serum samples was 61.1% (11/18) and 100% (11/11), respectively. However, mutantenriched sequencing identified an additional deletion in exon 19 and two L858R mutations. In this study, 21.4% (3/14) of the mutations could not be detected in serum samples by using non-enriched sequencing compared with mutant-enriched sequencing. Discussion This study demonstrated the possibility of using serum DNA as a surrogate to tissue for the assessment of EGFR mutation status. To our knowledge this is the first study to report that, in Chinese NSCLC patients, the EGFR mutation status determined in serum DNA using mutant-enriched sequencing corresponds to that demonstrated in paired tumour tissues (concordance rate of 93.1%), suggesting that serum DNA is a practical and reliable source of tumour DNA for detecting EGFR mutations. Several groups have already reported the use of other methods to detect serum EGFR 1396

6 TABLE 2: Clinical features of the patients with epidermal growth factor receptor (EGFR) gene mutations in serum and tumour tissue determined by mutant-enriched sequencing EGFR mutation status a Smoking Patient No. Sex Histology Stage history Tumour tissue Serum 1 F Adenocarcinoma IV Never E746_A750del E746_A750del 2 F Adenocarcinoma IIIB Never L858R 3 F Adenocarcinoma IV Never L858R L858R 4 F Adenocarcinoma IV Never L858R L858R 5 M Adenocarcinoma IIIB Current E746_A750del 6 F Adenocarcinoma IV Never L858R L858R 7 M Adenocarcinoma IIIB Current E746_A750del 8 M Adenocarcinoma IV Former E746_A750del E746_A750del 9 F Adenocarcinoma IV Never E746_A750del E746_A750del 10 F Adenocarcinoma IV Never L858R L858R 11 M Adenocarcinoma IIIB Current E746_A750del E746_A750del 12 F Adenocarcinoma IV Never L858R L858R 13 F Adenocarcinoma IV Never L858R L858R 14 M Adenocarcinoma IIIB Current E746_A750del E746_A750del 15 F Adenocarcinoma IV Never L858R 16 M Adenocarcinoma IV Former E746_A750del E746_A750del 17 F Adenocarcinoma IV Never E746_A750del E746_A750del 18 F Adenocarcinoma IV Never E746_A750del E746_A750del a, Indicates that no mutation was detected. F, female; M, male. 1397

7 TABLE 3: Association between clinical characteristics and epidermal growth factor receptor (EGFR) gene mutation status in serum and tumour tissue determined by mutant-enriched sequencing EGFR mutation status Tumour tissue Serum Clinical Wild Statistical Wild Statistical characteristics Mutation type significance Mutation type significance Sex, n P < P < Female Male Smoking history, n P = P = Never Current/former Histology, n P = P = Adenocarcinoma Non-adenocarcinoma mutations. For example, a study using the Scorpion Amplification Refractory Mutation System (ARMS ) showed a high consistency rate (92.9%) of EGFR mutation status in paired serum and tissue samples. 22 Another study using dhplc showed a high correlation between the mutations detected in plasma DNA and corresponding tumour DNA. 23 The high sensitivity (92%) and specificity (100%) of plasma EGFR mutation analysis by microfluidics digital PCR has also been reported. 26 Combining these findings with those of the present study, we believe that measuring EGFR mutations in serum may provide a feasible source of genotypic information in patients with tumours that are difficult to biopsy. For the detection of EGFR mutations, direct sequencing is commonly used as a first-line assay. 17 However, as direct sequencing can only detect mutant sequences constituting > 30% of the total genetic content, it is not useful for the detection of EGFR mutations in body fluids, in which only a small fraction of the EGFR sequences are mutated. 27 Additionally, lung cancers are very heterogeneous and the patient s serum also contains DNA derived from normal cells; thus, direct sequencing could miss a significant proportion of the mutations in these heterogeneous specimens. Therefore, mutant-enriched PCR-coupled sequencing, a rapid assay with high specificity and sensitivity that can detect one mutant gene among as many as copies of the wild-type gene, was used in the present study to provide mutational information that is more reliable. 25,28,29 As expected, the mutation status detected by mutantenriched sequencing in serum samples was nearly identical to that in tumour samples. In addition, it seems that mutant-enriched sequencing is more sensitive than the nonenriched assay for the analysis of serum samples. In the present study, most EGFR mutation sites in Chinese NSCLC patients were inframe deletions in exon 19 and the mis-sense L858R mutation in exon 21, which is consistent with previous studies from outside of China. 16,18,19,30 32 The comparison between mutation status and clinical 1398

8 characteristics in the present study also confirmed the finding in previous studies (including our previous studies) that EGFR mutations are frequently present in small subgroups of NSCLC patients, including never smokers, females and patients with adenocarcinoma histology. 19,33,34 Patients with squamous cell carcinoma and large-cell carcinoma have been found not to have the exon 19 and 21 mutations of EGFR shown in the present study for NSCLC. 33 Moreover, since only 58 patients were recruited in this study, larger samples will be needed to verify the relationships between EGFR mutation and different clinical characteristics. The mutation status in the tumour and serum sample pairs from four of the 58 patients (6.9%) did not match (mutation was detected in tumour but not serum samples) when mutant-enriched sequencing was used, which has also been observed in other studies using Scorpion ARMS and dhplc methods. 22,23 Although it has been reported that the amount of DNA extracted can decline during prolonged storage of serum samples, 34 this is not a potential explanation of this result for the present study because the DNA concentration of the serum samples, even after 4 years of storage, remained steady in the range of ng/µl, which far exceeds the detection minimum of 4 ng/µl. There are likely, therefore, to be other reasons for this discrepancy between tumour tissue and serum samples. As suggested by Wang et al., 35 a possible explanation of the inconsistency in mutation status may be heterogeneity of the genetic abnormalities in the tumours. If the parts of the tumour that carry mutations shed less DNA into the blood than other parts of the tumour, the levels of circulating tumourderived DNA in the serum sample might be reduced and subsequent analyses might miss the mutations in serum DNA. The present data, considered in the context of previous findings, provide strong evidence to suggest that mutant-enriched sequencing may provide a non-invasive and sensitive method for detecting EGFR mutation status in serum in patients with unresectable NSCLC. A larger study is required to evaluate further the consistency of mutation status assessed from tumours and serum samples in Chinese patients with advanced NSCLC. Acknowledgements This study was supported by grants from the National Natural Science Foundation of China ( and ), the Key Project of Science and Technology Commission of Shanghai Municipality (08JC , and 10JC ), the Shanghai Leading Academic Discipline Project (S30203), the Shanghai Municipal Education Committee (08YZ47) and the Science and Technology Commission of Shanghai Municipality (10JC ). Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 7 March 2011 Accepted subject to revision 11 March 2011 Revised accepted 2 June 2011 Copyright 2011 Field House Publishing LLP References 1 Jemal A, Siegel R, Ward E, et al: Cancer statistics, CA Cancer J Clin 2006; 56: Dempke WC, Suto T, Reck M: Targeted therapies for non-small cell lung cancer. Lung Cancer 2010; 67: Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et 1399

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