肺癌患者循环DNA中表皮生长因子受体（EGFR）突变的检测及其临床意义
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摘要
目的探讨直接测序法、单克隆基因测序法和高分辨率溶解曲线法在肺癌患者血浆循环DNA中EGFR突变检测的临床应用价值,并结合临床资料分析肺癌患者EGFR突变与临床病理特征之间的关系。
方法收集苏州大学附属第一医院及上海部分三甲医院96例肺癌患者的血浆以及其中59例相对应的肿瘤组织,提取组织及血浆循环DNA,采用直接测序法、单克隆基因测序法及高分辨率溶解曲线法检测EGFR基因19,21外显子的突变,并将高分辨率溶解曲线法检测的结果与直接测序法、单克隆基因测序法的结果进行了比较;同时应用高分辨率溶解曲线法检测含不同比例EGFR基因突变型质粒的系列混合样本,评价灵敏度。
结果
1. 96例肺癌血浆中检测出EGFR突变17例,其突变率为17.7%。在这些突变的样本中,外显子19和21突变分别占88.2%(15/17例)和11.8%(2/17例),其中直接测序法检测出EGFR纯合突变3例(L858R 2例、del E746-A750(1)1例),杂合突变14例。对这14例杂合突变样本进一步通过单克隆基因测序法确定了它们的突变类型为(del E746-A750(2)9例,del E746- A750(1)3例,del L747-S752 2例)。
2.经HRM法,在96例肺癌患者中检测出17例发生了EGFR突变,突变率为17.7%,其中外显子19和21突变分别占88.2%(15/17例)和11.8%(2/17例);经与测序法的结果比对完全一致。此外,HRM法可检出系列混合样本中突变型质粒比例为5%的突变,其检测灵敏度达5%。
3. EGFR基因突变多见于肺腺癌患者,与患者的性别与吸烟史无明显相关性。对59例肺癌患者肿瘤组织的进一步分析证明,血浆EGFR基因突变类型与患者自身肿瘤的突变类型相同,表明血浆DNA检测到的EGFR突变与原发肿瘤检测到的突变相一致。
结论
1.传统的直接测序法可用于基因突变的检测,但当检测到杂合突变样本时,由于其测序图谱出现杂合套峰,不能确定样本的突变类型,而此时通过单克隆基因测序法就能明确地判断基因的突变类型。
2. HRM法可作为临床EGFR突变筛查的优选方法,其操作简单易行,快速,灵敏度为5%,与测序法比较,检测结果一致。
3.肺癌患者的血浆循环DNA与相对应的肿瘤组织DNA的EGFR基因突变类型一致,可说明以血浆游离DNA可作为肿瘤组织DNA的替代材料进行EGFR基因突变检测。
Purpose: This study aims to analyze the clinical value of epidermal growth factor receptor (EGFR) mutation detection among direct sequencing, monoclonal sequencing and high resolution melting curve analysis (HRM) in the plasma circulating DNA of lung cancer patients. In addition, this study also discusses the relation between EGFR mutations and clinical pathological features with analysis of the clinical data.
Method: The 96 plasma samples of lung cancer patients were from The First Affiliated Hospital of Soochow University and some upper first-class hospitals in Shanghai, 59 of which had corresponding tumor tissues. The DNA were extracted from the samples and the mutations of EGFR’s exon19 and 21 were detected by direct sequencing, monoclonal sequencing and HRM. The results of HRM were compared to that of direct sequencing and monoclonal sequencing. The sensitivity of HRM was analyzed by the detection of samples containing different proportions of EGFR-mutated plasmids.
Results:
1. Somatic mutations of EGFR were identified in plasma from 17 of the 96 cases(17. 7%), including 15 cases (88.2%) in exon 19 and 2 cases (11.8%) in exon 21. Among all the 17 detected mutation samples, 3 homozygous mutation (2 L858R samples, 1 del E746-A750 (2) sample) and 14 heterozygous mutation samples are detected by direct sequencing. Then monoclonal sequencing is used to further determine the types of mutation in those heterozygous mutation samples mentioned above (9 del E746-A750(2) samples, 3 del E746-A750 (1) samples, 2 del L747-S752 samples).
2. By means of HRM within 96 lung cancer patients, EGFR mutations were detected in 17(17/96,17.7%) cases, in which the number of cases of exon 19 and 21 mutations is 15 (15/17,88.2%) and 2 (2/17, 11.8%) respectively. The results of sequencing are consistent with it. HRM detection could identify EGFR mutation in a proportion of 5% of mutated plasmid DNA.
3. Patients with adenocarcinoma had an increased frequency of mutations.The mutation was not closely related with gender and smoking history.Further analysis on 59 lung cancer patients demonstrated that the EGFR mutation in plasma was consistent with that detected in tumor tissues, suggesting that the EGFR mutations in plasma originated from the primary tumor.
Conclusion
1. Traditional method of direct sequencing could be applied to the detection of gene mutation. However, when sequencing the heterozygous samples, it cannot determine the types of mutations according to sequencing plots because of the heterozygous peaks. But by means of monoclonal sequencing, the plots of it have clean backgrounds and easy to determine the mutation types.
2. The HRM analysis could be an optimal method for clinical screening of EGFR mutation thanks to its simpleness, swiftness and high sensitiveness to 5% mutation in the samples. Furthermore, the results of HRM are identical with that of sequencing.
3. The EGFR mutations in plasma circulating DNA are in accordance with that in lung cancer tissues, demonstrating that plasma circulating DNA could be applied to the mutation detection in replace of tissue DNA.
方法收集苏州大学附属第一医院及上海部分三甲医院96例肺癌患者的血浆以及其中59例相对应的肿瘤组织,提取组织及血浆循环DNA,采用直接测序法、单克隆基因测序法及高分辨率溶解曲线法检测EGFR基因19,21外显子的突变,并将高分辨率溶解曲线法检测的结果与直接测序法、单克隆基因测序法的结果进行了比较;同时应用高分辨率溶解曲线法检测含不同比例EGFR基因突变型质粒的系列混合样本,评价灵敏度。
结果
1. 96例肺癌血浆中检测出EGFR突变17例,其突变率为17.7%。在这些突变的样本中,外显子19和21突变分别占88.2%(15/17例)和11.8%(2/17例),其中直接测序法检测出EGFR纯合突变3例(L858R 2例、del E746-A750(1)1例),杂合突变14例。对这14例杂合突变样本进一步通过单克隆基因测序法确定了它们的突变类型为(del E746-A750(2)9例,del E746- A750(1)3例,del L747-S752 2例)。
2.经HRM法,在96例肺癌患者中检测出17例发生了EGFR突变,突变率为17.7%,其中外显子19和21突变分别占88.2%(15/17例)和11.8%(2/17例);经与测序法的结果比对完全一致。此外,HRM法可检出系列混合样本中突变型质粒比例为5%的突变,其检测灵敏度达5%。
3. EGFR基因突变多见于肺腺癌患者,与患者的性别与吸烟史无明显相关性。对59例肺癌患者肿瘤组织的进一步分析证明,血浆EGFR基因突变类型与患者自身肿瘤的突变类型相同,表明血浆DNA检测到的EGFR突变与原发肿瘤检测到的突变相一致。
结论
1.传统的直接测序法可用于基因突变的检测,但当检测到杂合突变样本时,由于其测序图谱出现杂合套峰,不能确定样本的突变类型,而此时通过单克隆基因测序法就能明确地判断基因的突变类型。
2. HRM法可作为临床EGFR突变筛查的优选方法,其操作简单易行,快速,灵敏度为5%,与测序法比较,检测结果一致。
3.肺癌患者的血浆循环DNA与相对应的肿瘤组织DNA的EGFR基因突变类型一致,可说明以血浆游离DNA可作为肿瘤组织DNA的替代材料进行EGFR基因突变检测。
Purpose: This study aims to analyze the clinical value of epidermal growth factor receptor (EGFR) mutation detection among direct sequencing, monoclonal sequencing and high resolution melting curve analysis (HRM) in the plasma circulating DNA of lung cancer patients. In addition, this study also discusses the relation between EGFR mutations and clinical pathological features with analysis of the clinical data.
Method: The 96 plasma samples of lung cancer patients were from The First Affiliated Hospital of Soochow University and some upper first-class hospitals in Shanghai, 59 of which had corresponding tumor tissues. The DNA were extracted from the samples and the mutations of EGFR’s exon19 and 21 were detected by direct sequencing, monoclonal sequencing and HRM. The results of HRM were compared to that of direct sequencing and monoclonal sequencing. The sensitivity of HRM was analyzed by the detection of samples containing different proportions of EGFR-mutated plasmids.
Results:
1. Somatic mutations of EGFR were identified in plasma from 17 of the 96 cases(17. 7%), including 15 cases (88.2%) in exon 19 and 2 cases (11.8%) in exon 21. Among all the 17 detected mutation samples, 3 homozygous mutation (2 L858R samples, 1 del E746-A750 (2) sample) and 14 heterozygous mutation samples are detected by direct sequencing. Then monoclonal sequencing is used to further determine the types of mutation in those heterozygous mutation samples mentioned above (9 del E746-A750(2) samples, 3 del E746-A750 (1) samples, 2 del L747-S752 samples).
2. By means of HRM within 96 lung cancer patients, EGFR mutations were detected in 17(17/96,17.7%) cases, in which the number of cases of exon 19 and 21 mutations is 15 (15/17,88.2%) and 2 (2/17, 11.8%) respectively. The results of sequencing are consistent with it. HRM detection could identify EGFR mutation in a proportion of 5% of mutated plasmid DNA.
3. Patients with adenocarcinoma had an increased frequency of mutations.The mutation was not closely related with gender and smoking history.Further analysis on 59 lung cancer patients demonstrated that the EGFR mutation in plasma was consistent with that detected in tumor tissues, suggesting that the EGFR mutations in plasma originated from the primary tumor.
Conclusion
1. Traditional method of direct sequencing could be applied to the detection of gene mutation. However, when sequencing the heterozygous samples, it cannot determine the types of mutations according to sequencing plots because of the heterozygous peaks. But by means of monoclonal sequencing, the plots of it have clean backgrounds and easy to determine the mutation types.
2. The HRM analysis could be an optimal method for clinical screening of EGFR mutation thanks to its simpleness, swiftness and high sensitiveness to 5% mutation in the samples. Furthermore, the results of HRM are identical with that of sequencing.
3. The EGFR mutations in plasma circulating DNA are in accordance with that in lung cancer tissues, demonstrating that plasma circulating DNA could be applied to the mutation detection in replace of tissue DNA.
引文
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[3] Paez JG, Janne PA, Lee JC, et a1. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy[J]. Science. 2004, 304(5676):1497-1500.
[4] Huang SF, Liu HP, Li LH, et a1. High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in taiwan[J]. Clin Cancer Res. 2004, 10(24):8l95-8203.
[5] Pao W, Miller V, Zakowski M, et a1. EGF receptor gene mutations are common in lung cancers from“never smokers”and are associated with sensitivity of tumors to gefitinib and erlotinib[J]. Proc Natl Acad Sci USA. 2004, 101(36):13306-13311.
[6] Gazdar AF, Shigematsu H, Herz J, et a1. Mutations and addiction to EGFR :The achilles“heal”of lung cancers[J]. Trends Mol Med. 2004, 10(10):481-486.
[7] Pao W, Miller VA. Epidermal growth factor receptor mutations,small-molecule kinase inhibitors,and non-small-cell lung cancer:Curent knowledge and future directions[J]. J Clin Oncol. 2005, 23(11):2556-2568.
[8] Tam IYS, Chung LP, Suen WS, et a1. Distinct epidermal growth factor receptor and KRAS mutation patterns in non-small cell lung cancer patients with diferent to bacco exposure and clinicopathologic features[J]. Clin Cancer Res. 2006, 12(5):1647-1653.
[9] Stroun M, Maurice P, Vasioukhin V, et al. The origin and mechanism of circulating DNA[J]. Ann NY Acad Sci. 2000, 906:161-168.
[10] Stroun M, Lyautey J, Lederrey C, et al. About the possible origin and mechanism ofcirculating DNA apoptosis and active DNA release[J]. Clin Chim Acta. 2001, 313: 139-142.
[11] Yoon KA, Park S, Lee SH, et al. Comparison of circulating plasma DNA levels between lung cancer patients and healthy controls[J]. J Mol Diagn. 2009, 11(3):182- 185.
[12] Paci M, Maramotti S, Bellesia E,et al. Circulating plasma DNA as diagnostic biomarker in non-small cell lung cancer[J]. Lung Cancer. 2009, 64(1):92-97.
[13] Maheswaran S, Sequist LV, Nagrath S, et al. Detection of mutations in EGFR in circulating lung-cancer cells[J]. N Engl J Med. 2008, 359 (4):366-377.
[14] Frank Diehl, Kerstin Schmidt, Michael A Choti, et al. Circulating mutant DNA to assess tumor dynamics[J]. Nature Medicine. 2008, 9(14):985-990.
[15] Gow CH, ChangYL, Hsu YC, et a1. Comparison of epidermal growth factor receptor mutations between primary and corresponding metastatic tumors in tyrosine kinase inhibitor-naive non-small-cell lung cancer[J]. Ann Oncol. 2009, 20(4):696-702.
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