DHPLC检测多种途径获取的NSCLC组织EGFR基因突变及NSCLC原发病灶与配对淋巴结转移灶中EGFR/K-ras基因型的比较
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摘要
肺癌是目前全球死亡率最高的恶性肿瘤,因为缺乏特异性症状及有效的筛查手段,仅16%患者初诊时为早期,其中大多数为非小细胞肺癌(non-small cell lung cancer,NSCLC)。目前NSCLC的治疗效果难以令人满意,现主要的治疗手段包括手术、放疗、化疗、靶向治疗等,而手术是唯一的根治手段。患者表皮生长因子受体(epidermal growth factor receptor, EGFR)酪氨酸激酶区(tyrosine kinase, TK)已成为目前研究进展最快,最受瞩目的NSCLC治疗靶点,吉非替尼、厄罗替尼作为常见的酪氨酸激酶抑制剂(tyrosine kinase inhibitors, TKIs),已广泛应用于治疗NSCLC晚期病人。TKIs的疗效与EGFR TK区是否存在突变以及突变类型有关。K-ras是RAS家族的重要成员,K-ras突变与EGFR TKIs的原发耐药相关,同时K-ras突变与NSCLC患者预后不良密切相关。据此,在对晚期NSCLC患者进行治疗决策前,明确其EGFR/K-ras基因型是非常必要的。目前,DNA直接测序是检测EGFR突变最常用的方法,同时也作为突变检测的“金标准”,但该方法步骤繁琐、耗时较长、费用较高、所需组织量较多且敏感度较低,并不能满足临床需求。变性高效液相色谱(denaturing high performance liquid chromatography, DHPLC)是一种新的高通量筛选基因序列变异的技术,其原理是利用离子对反向高效液相色谱法在部分变性的温度条件下分离、识别野生型及突变型DNA双链,具有快速、自动化、敏感性、特异性高等特点,可用于基因突变的检测。TKIs已在晚期EGFR敏感性突变的NSCLC患者的治疗中获得巨大的成功,应用TKIs进行可手术患者的诱导治疗已成为新的探索模式。恶性肿瘤是异质性明显的疾病,众多研究发现同一NSCLC患者的原发病灶及转移灶之间EGFR/K-ras基因状态也存在较大的差异。本研究尝试探讨DHPLC'快速检测由多种途径获取的NSCLC肿瘤组织EGFR基因突变的诊断价值,此外比较了非小细胞肺癌原发灶与配对淋巴结转移灶中EGFR/K-ras基因型的相关性。
本研究分为两部分。
第一部分：DHPLC检测多种途径获取的NSCLC组织EGFR基因突变
目的：探讨DHPLC快速检测由CT引导经皮细针肺穿刺活检、淋巴结活检以及外科切除三种途径获取的NSCLC肿瘤组织EGFR基因突变的诊断价值,并分析EGFR突变与患者临床特征之间的相关性。
方法：
1.选取2008年1月至2009年6月广东省人民医院收治的83位非小细胞肺癌患者的肿瘤组织,其中CT引导经皮细针肺穿刺活检标本37份、淋巴结活检标本15份、以及外科切除标本31份。所有肿瘤标本离体后等分为二,一部分以10%中性福尔马林固定用于石蜡切片、病理诊断,另一部分立即置入液氮中,后转入-80℃低温冰箱中保存备用。所有患者获取标本前均未接受化疗或靶向治疗,相应组织经病理确诊后纳入研究。
2.组织DNA的提取：组织匀浆法提取肿瘤组织中的总DNA,经Eppendorf核酸蛋白测定仪测定DNA纯度及含量。
3.采用TA克隆法分别构建EGFR基因19外显子、21外显子突变型(del746-750、L858R)和野生型质粒,分别按1：1、1：5、1：10、1：20、1：50、1：100比例将突变型、野生型混合,用于直接测序法、DHPLC法检测敏感性分析。
4.PCR扩增EGFR基因19、21外显子的基因片段,2%琼脂糖凝胶电泳鉴定扩增片段。PCR产物分别用作直接测序法及DHPLC法检测。
5.PCR产物切胶过柱纯化后使用ABI3100测序仪进行测序检测,采用Chromas软件分析测序图谱。
6.PCR产物不作任何纯化处理,直接用于DHPLC分析。检测敏感性分析中EGFR19外显子分别采用非变性条件及部分变性条件进行检测,并进行比较；21外显子采用部分变性条件进行检测。对肿瘤组织EGFR19外显子、21外显子均采用部分变性条件进行检测。
7.统计学分析：采用SPSS 13.0软件进行统计学分析。应用χ2检验比较EGFR突变与患者性别、吸烟状态、病理类型的关系。应用两独立样本t检验比较EGFR突变型与野生型患者之间年龄是否存在差异。以P<0.05认为差异有统计学意义。
结果：
1. DHPLC检测的敏感性：当突变型质粒与野生型质粒按1：100混合时,仍可被DHPLC法显著检出,而直接测序法仅可检出1：10水平。非变性条件及部分变性条件检测EGFR 19外显子敏感性相似。
2.83例NSCLC组织标本中,DHPLC法检出22例EGFR突变(突变率26.51%),3例直接测序法结果为野生型,余19例EGFR突变及61例野生型均与直接测序法结果相符。以直接测序法为参考标准,DHPLC法的敏感度为100%,特异度为95.31%,检测对经皮细针肺穿刺活检、淋巴结活检以及外科切除等途径获取的肿瘤样本的敏感度均为100%,特异度分别为96.55%、100.00%及92.00%。
3.以DHPLC法为参考标准,直接测序法的敏感度为86.36%。
4.女性患者EGFR突变率(37.93%)高于男性患者(突变率14.81%,χ2=5.712,P=0.017),腺癌患者EGFR突变率(32.14%)高于非腺癌患者(突变率3.70%,χ2=8.347,P=0.004),差异均有统计学意义；符合女性、非吸烟、腺癌的NSCLC患者EGFR突变率达40.74%。但EGFR突变与患者吸烟状态无显著相关(P>0.05)。
结论：
本研究发现,DHPLC法具有很高的检测敏感性,部分变性条件也适用于EGFR 19外显子检测,而且有利于发现未知突变。DHPLC作为EGFR突变检测的初筛方法,与“金标准”具有高度的一致性。同时本研究中肿瘤样本分别来源于经皮肺穿刺、淋巴结活检以及外科手术切除三种临床最常用的途径,DHPLC均获得与直接测序法高度一致的结果,显示该方法对不同类型的标本均可有效地进行突变检测。同时本研究结果提示83例NSCLC患者EGFR突变率为22.89%,女性、腺癌患者中EGFR突变率显著高于男性、非腺癌患者,EGFR突变与吸烟状态、年龄等临床特征无显著相关。综上所述,DHPLC法可作为NSCLC患者EGFR基因型的初筛方法。
第二部分：非小细胞肺癌原发灶与配对淋巴结转移灶中EGFR/K-ras基因型的比较
目的：探讨非小细胞肺癌原发灶与配对淋巴结转移灶之间EGFR/K-ras基因型的相关性。
方法
1.选取2007年5月至2010年2月广东省人民医院收治的21位非小细胞肺癌患者的原发病灶及淋巴结转移灶肿瘤组织。其中原发病灶取自手术切除者14例,取自CT引导下经皮肺穿刺者7例；淋巴结病灶取自手术切除者7例,取自纵隔镜活检者11例,取自锁骨下淋巴结活检术者3例。所有肿瘤标本离体后均分为二,一部分使用10%中性福尔马林固定送广东省人民医院病理科行常规病理诊断；一部分立即投入液氮中冻存,后转入-80°超低温冰箱中备用。
2.组织DNA的提取：组织匀浆法提取肿瘤组织中的总DNA,经Eppendorf核酸蛋白测定仪测定DNA纯度及含量。
3.PCR扩增EGFR基因18-21外显子和K-ras12、13密码子以及59密码子的基因片段,2%琼脂糖凝胶电泳鉴定扩增片段。PCR产物用作直接测序。
4.PCR产物切胶过柱纯化后使用ABI3100测序仪进行测序检测,采用Chromas软件分析测序图谱,突变DNA阳性结果再通过反向引物测序进行验证。
5.统计学分析：采用SPSS 13.0软件进行统计学分析。应用Kappa一致性检验比较NSCLC原发病灶及配对的淋巴结转移灶之间EGFR/K-ras基因型的一致性,以P<0.05认为一致性有统计学意义,并参考Kappa系数评价标准,当Kappa系数<0,认为一致性强度极差；0.0-0.2,微弱；0.21-0.40,弱；0.41-0.60,中度；0.61-0.80,高度；0.81-1.00,极强。
结果：
1.21例患者中仅发现1例K-ras G59G突变(突变率为4.76%),且NSCLC原发灶及淋巴结转移灶突变类型一致；检出NSCLC原发病灶与淋巴结转移病灶存在相同EGFR突变患者6例,其中EGFR 21外显子L858R突变者4例,21外显子L858R及20外显子T790M突变1例,19外显子缺失型突变1例；原发灶与淋巴结转移灶EGFR基因型不一致者1例,原发病灶为EGFR野生型、淋巴结转移灶为EGFR 19外显子缺失；原发灶、淋巴结转移灶EGFR/K-ras均是野生型者13例。
2.本研究发现,NSCLC原发病灶与配对淋巴结转移灶的EGFR/K-ras基因型一致(EGFR/K-ras基因Kappa系数分别为0.889及1.000,P<0.001)。
结论：采用新鲜肿瘤组织进行检测,无论是同时或异时获取,同一患者配对的NSCLC原发病灶和区域淋巴结转移灶间EGFR/K-ras基因状态基本一致。但有待扩大样本量进一步研究。
Lung cancer is a major cause of cancer related mortality worldwide. The clinical manifestations and Early Detection of lung cancer are lack, only 16% lung cancer patients are diagosed in early stage, most of these cases are non-small-cell lung cancer (NSCLC). The main therapeutic methods include surgery, radiotherapy, chemotherapy and targeted therapy, among which operation is the only radical method, but the therapeutic effect is disappointed.Epidermal growth factor receptor(EGFR) tyrosine kinase domain has become the fastest developing Therapeutic target now.Gefitinib and erlotinib are the most common tyrosine kinase inhibitors, and have been widely used in the treatment of patients with advanced NSCLC. The efficacy of TKIs is associated with EGFR mutation. K-ras is one of the most important members of RAS family. K-ras mutation may leading to the primary resistance of EGFR TKIs and poor prognosis of NSCLC patients. It is very necessary to comform the EGFR gene type before the treatment of patients suffering from advanced NSCLC. Currently, direct sequencing is the most common method of detection EGFR mutation, and is also as the "gold standard". But direct sequencing is complex, time-and cost-intensive, less sensitive. As a result, direct sequencing isn't able to fulfill the requirement. The Denaturing High-Performance Liquid Chromatography(DHPLC) is a new technology of high-throughput screening of gene variations.wild type and mutant DNA were detected and separated in partial denaturing condition by ion-pair-reverse-phase liquid chromatography.DHPLC is a rapid automated sensitive and specific method in mutant gene detection. TKIs had be successful in the treatment of advanced NSCLC.The neoadjuvant TKIs in operable NSCLC patients would be a new treatment model. But the heterogeneity of malignant tumors is significant. Many studies showed discrepancy in EGFR mutation between primary tumors and corresponding metastasis. We evaluated that DHPLC as a rapid detection method for EGFR mutation in NSCLC tumor specimens and compared the EGFR and K-ras genotypes between primary tumors and corresponding metastatic lymph node in NSCLC.
This research consists two parts
Part one
Detection of EGFR mutations in NSCLC tumor specimens from various ways by DHPLC and comparison of EGFR
Objective
Evaluation DHPLC as a rapid detection method for EGFR mutations of NSCLC tumor specimens from CT guided transthoracic needle biopsy of lung, lymph node biopsy and surgical resection. Correlation analysis of EGFR mutations and clinical characteristic of NSCLC patients.
Method
1.83 NSCLC patients hospitalized in guangdong general hospital from January 2008 to June 2009 were included. There were 37 tumor specimens from CT guided transthoracic needle biopsy of lung,15 from Lymph Node Biopsy, and 31 from surgical resection. The tumor specimens were divided into two, a section fixed by 10% neutral formalin and paraffin embedded was used for pathological diagnosis. The other section was put into liquid nitrogen and then transferred to -80℃low temperature refrigerator. All the patients were naive chemotherapy or targeting therapy, the specimens included in the study have be pathologically confirmed.
2. DNA extraction:The genomic DNA was extracted from lung cancer tissues.The DNA purity and content were detected by the Eppendorf nucleic acid protein cryoscope.
3. TA cloning method was used to constructed EGFR gene exon 19, exon 21 wild-type and mutant (del746-750, L858R) plasma. The 1:1,1:5,1:10,1:20,1:50,1:100 dilutions of mutant and wild type EGFR plasma DNA were using in evaluation the sensitivity of DHPLC and direct sequencing.
4. The EGFR exon 19,21 were amplified by PCR. PCR products were used for direct sequencing and DHPLC detection after being evaluated by 2% agarose gel electrophoresis.
5. The PCR products were purified.Direct sequencing of the PCR products was performed using ABI PRISM 3100 DNA Analyzer. Chromas software was used to analyze the sequencing results.
6. PCR products were detected by DHPLC without any purification. We compared the sensitivity of non-denatured condition and the partial denatured condition in the detection of the EGFR exon 19. Exon 21 was evaluated by partial denatured condition. The exon 19,21 were detected by partial denatured condition in tumor specimens.
7. Statistics analysis:All data were analyzed with SPSS 13.0 software. Chi-square test was used in examination of the relationship between EGFR mutations and gemder, smoking status, pathological type. P< 0.05 was considered statistically significant for all analyses.
Result
1. evaluation of DHPLC sensitivity:Mutant plasma DNA can be detected in the serial dilution of 1:100 by DHPLC and 1:10 by direct sequencing. The sensitivity of detecting EGFR exon 19 is similar in the condition of non-denaturing and partial denaturing.
2. DHPLC showed 22 EGFR mutations in 83 NSCLC patients,19 samples had been conformed by direct sequencing except 3 samples, the other 61 samples are wild type by DHPLC and direct sequencing. Compared with direct sequencing, the sensitivity of DHPLC was 100%, and the specificity was 95.31%.The sensitivity of detection tumor specimens from CT-guided transthoracic needle lung biopsy, lymph node Biopsy and surgical resection were 100%,and the specificity of detection tumor specimens from CT-guided transthoracic needle lung biopsy, lymph node Biopsy and surgical resection were 96.55%,100.00% and 92.00% respectively.
3. Compared with DHPLC the sensitivity of direct sequencing was 86.36%.
4. EGFR mutations were present in 37.93% female and 14.81% male(x2=5.712, P=0.017);in 32.14% adenocarcinoma and 3.70% non-adenocarcinoma(x2=8.347, P=0.004), There was no statistical difference. EGFR mutations were present in 40.74%.Feminine, non-smoking, the adenocarcinoma NSCLC patients. But the EGFR genotype was not associated with smoking status (P>0.05).
Conclusion
DHPLC is a sensitive method for screening EGFR mutations, partial denatured condition is applicable to detecting EGFR exon 19, and can detect unknown mutation. DHPLC is in accordance with the "gold standard" and is a precise rapid preliminary screening method for detection of NSCLC EGFR genotype. The tumor specimens were from CT-guided transthoracic needle lung biopsy, lymph node Biopsy and surgical resection which are the most commonly ways, the results of DHPLC is in accordance with direct sequencing. EGFR mutations were present in 22.89% of 83 NSCLC patients.Female and adenocarcinoma patients showed higher mutation frequency. Smoking status and age were no significant correlation with EGFR mutations. In conclusion DHPLC is a precise rapid preliminary screening method for detection of NSCLC EGFR genotype.
Part two Comparison of EGFR and K-ras genotypes between primary tumors and corresponding lymph node Metastases in NSCLC
Objective
Correlation analysis of EGFR and K-ras genotypes between primary tumors and corresponding metastatic lymph node in NSCLC.
Method
1. Macthed primary tumors and metastatic lymph nodes in 21 NSCLC patients hospitalized in guangdong general hospital from may 2008 to February 2010 were included. There were 14 primary tumor specimens from surgical resection and the other 7 from CT guided transthoracic needle biopsy of lung. There were 7 metastatic lymph nodes from surgical resection,11 from Mediastinoscopy and the other 3 from infraclavicula lymph node biopsy. The tumor specimens were divided into two, a section fixed by 10% neutral formalin and paraffin embedded was used for pathological diagnosis. The other section was put into liquid nitrogen and then transferred to-80℃low temperature refrigerator.
2. DNA extraction:The genomic DNA was extracted from lung cancer tissues.The DNA purity and content were detected by the Eppendorf nucleic acid protein cryoscope.
3. The EGFR exon 18-21 and K-ras codon 12,13,59 were amplified by PCR. PCR products were used for direct sequencing and DHPLC detection after being evaluated by 2% agarose gel electrophoresis.
4. The PCR products were purified.Direct sequencing of the PCR products was performed using ABI PRISM 3100 DNA Analyzer. Chromas software was used to analyze the sequencing results.
5. Statistics analysis:All data were analyzed with SPSS 13.0 software. Kappa test was used in examination of the homogeneity in EGFR and K-ras genotypes between primary tumors and corresponding metastatic lymph node in NSCLC. P< 0.05 was considered statistically significant for homogeneity. Examination of the homogeneity combined the Kappa value.
Result
A K-ras mutation was present in 4.76% of 21 patients, and was concordant in primary tumors and corresponding metastatic lymph node. We found concordant EGFR mutations in 6 patients.4 cases harbored exon 21 L858R mutation,1 case harbored exon 21 L858R and exon 20 T790M mutations,1 case harbored exon 19 delection. Discordant EGFR mutation was present in only one case, primary tumor harbored EGFR wild type, but metastatic lymph node harbored EGFR exon 19 delection. Concordant EGFR and K-ras wild type were found in 13 patients. EGFR and K-ras genotypes of primary tumors and corresponding metastatic lymph node in , NSCLC were concordant(Kappa value of EGFR and K-ras is 0.889 and 1.000, P<0.001).
Conclusion
EGFR and K-ras genotypes of primary tumors and corresponding metastatic lymph node in NSCLC were concordant by detecting fresh tissues, but we need enlarge the sample size for further research.
本研究分为两部分。
第一部分：DHPLC检测多种途径获取的NSCLC组织EGFR基因突变
目的：探讨DHPLC快速检测由CT引导经皮细针肺穿刺活检、淋巴结活检以及外科切除三种途径获取的NSCLC肿瘤组织EGFR基因突变的诊断价值,并分析EGFR突变与患者临床特征之间的相关性。
方法：
1.选取2008年1月至2009年6月广东省人民医院收治的83位非小细胞肺癌患者的肿瘤组织,其中CT引导经皮细针肺穿刺活检标本37份、淋巴结活检标本15份、以及外科切除标本31份。所有肿瘤标本离体后等分为二,一部分以10%中性福尔马林固定用于石蜡切片、病理诊断,另一部分立即置入液氮中,后转入-80℃低温冰箱中保存备用。所有患者获取标本前均未接受化疗或靶向治疗,相应组织经病理确诊后纳入研究。
2.组织DNA的提取：组织匀浆法提取肿瘤组织中的总DNA,经Eppendorf核酸蛋白测定仪测定DNA纯度及含量。
3.采用TA克隆法分别构建EGFR基因19外显子、21外显子突变型(del746-750、L858R)和野生型质粒,分别按1：1、1：5、1：10、1：20、1：50、1：100比例将突变型、野生型混合,用于直接测序法、DHPLC法检测敏感性分析。
4.PCR扩增EGFR基因19、21外显子的基因片段,2%琼脂糖凝胶电泳鉴定扩增片段。PCR产物分别用作直接测序法及DHPLC法检测。
5.PCR产物切胶过柱纯化后使用ABI3100测序仪进行测序检测,采用Chromas软件分析测序图谱。
6.PCR产物不作任何纯化处理,直接用于DHPLC分析。检测敏感性分析中EGFR19外显子分别采用非变性条件及部分变性条件进行检测,并进行比较；21外显子采用部分变性条件进行检测。对肿瘤组织EGFR19外显子、21外显子均采用部分变性条件进行检测。
7.统计学分析：采用SPSS 13.0软件进行统计学分析。应用χ2检验比较EGFR突变与患者性别、吸烟状态、病理类型的关系。应用两独立样本t检验比较EGFR突变型与野生型患者之间年龄是否存在差异。以P<0.05认为差异有统计学意义。
结果：
1. DHPLC检测的敏感性：当突变型质粒与野生型质粒按1：100混合时,仍可被DHPLC法显著检出,而直接测序法仅可检出1：10水平。非变性条件及部分变性条件检测EGFR 19外显子敏感性相似。
2.83例NSCLC组织标本中,DHPLC法检出22例EGFR突变(突变率26.51%),3例直接测序法结果为野生型,余19例EGFR突变及61例野生型均与直接测序法结果相符。以直接测序法为参考标准,DHPLC法的敏感度为100%,特异度为95.31%,检测对经皮细针肺穿刺活检、淋巴结活检以及外科切除等途径获取的肿瘤样本的敏感度均为100%,特异度分别为96.55%、100.00%及92.00%。
3.以DHPLC法为参考标准,直接测序法的敏感度为86.36%。
4.女性患者EGFR突变率(37.93%)高于男性患者(突变率14.81%,χ2=5.712,P=0.017),腺癌患者EGFR突变率(32.14%)高于非腺癌患者(突变率3.70%,χ2=8.347,P=0.004),差异均有统计学意义；符合女性、非吸烟、腺癌的NSCLC患者EGFR突变率达40.74%。但EGFR突变与患者吸烟状态无显著相关(P>0.05)。
结论：
本研究发现,DHPLC法具有很高的检测敏感性,部分变性条件也适用于EGFR 19外显子检测,而且有利于发现未知突变。DHPLC作为EGFR突变检测的初筛方法,与“金标准”具有高度的一致性。同时本研究中肿瘤样本分别来源于经皮肺穿刺、淋巴结活检以及外科手术切除三种临床最常用的途径,DHPLC均获得与直接测序法高度一致的结果,显示该方法对不同类型的标本均可有效地进行突变检测。同时本研究结果提示83例NSCLC患者EGFR突变率为22.89%,女性、腺癌患者中EGFR突变率显著高于男性、非腺癌患者,EGFR突变与吸烟状态、年龄等临床特征无显著相关。综上所述,DHPLC法可作为NSCLC患者EGFR基因型的初筛方法。
第二部分：非小细胞肺癌原发灶与配对淋巴结转移灶中EGFR/K-ras基因型的比较
目的：探讨非小细胞肺癌原发灶与配对淋巴结转移灶之间EGFR/K-ras基因型的相关性。
方法
1.选取2007年5月至2010年2月广东省人民医院收治的21位非小细胞肺癌患者的原发病灶及淋巴结转移灶肿瘤组织。其中原发病灶取自手术切除者14例,取自CT引导下经皮肺穿刺者7例；淋巴结病灶取自手术切除者7例,取自纵隔镜活检者11例,取自锁骨下淋巴结活检术者3例。所有肿瘤标本离体后均分为二,一部分使用10%中性福尔马林固定送广东省人民医院病理科行常规病理诊断；一部分立即投入液氮中冻存,后转入-80°超低温冰箱中备用。
2.组织DNA的提取：组织匀浆法提取肿瘤组织中的总DNA,经Eppendorf核酸蛋白测定仪测定DNA纯度及含量。
3.PCR扩增EGFR基因18-21外显子和K-ras12、13密码子以及59密码子的基因片段,2%琼脂糖凝胶电泳鉴定扩增片段。PCR产物用作直接测序。
4.PCR产物切胶过柱纯化后使用ABI3100测序仪进行测序检测,采用Chromas软件分析测序图谱,突变DNA阳性结果再通过反向引物测序进行验证。
5.统计学分析：采用SPSS 13.0软件进行统计学分析。应用Kappa一致性检验比较NSCLC原发病灶及配对的淋巴结转移灶之间EGFR/K-ras基因型的一致性,以P<0.05认为一致性有统计学意义,并参考Kappa系数评价标准,当Kappa系数<0,认为一致性强度极差；0.0-0.2,微弱；0.21-0.40,弱；0.41-0.60,中度；0.61-0.80,高度；0.81-1.00,极强。
结果：
1.21例患者中仅发现1例K-ras G59G突变(突变率为4.76%),且NSCLC原发灶及淋巴结转移灶突变类型一致；检出NSCLC原发病灶与淋巴结转移病灶存在相同EGFR突变患者6例,其中EGFR 21外显子L858R突变者4例,21外显子L858R及20外显子T790M突变1例,19外显子缺失型突变1例；原发灶与淋巴结转移灶EGFR基因型不一致者1例,原发病灶为EGFR野生型、淋巴结转移灶为EGFR 19外显子缺失；原发灶、淋巴结转移灶EGFR/K-ras均是野生型者13例。
2.本研究发现,NSCLC原发病灶与配对淋巴结转移灶的EGFR/K-ras基因型一致(EGFR/K-ras基因Kappa系数分别为0.889及1.000,P<0.001)。
结论：采用新鲜肿瘤组织进行检测,无论是同时或异时获取,同一患者配对的NSCLC原发病灶和区域淋巴结转移灶间EGFR/K-ras基因状态基本一致。但有待扩大样本量进一步研究。
Lung cancer is a major cause of cancer related mortality worldwide. The clinical manifestations and Early Detection of lung cancer are lack, only 16% lung cancer patients are diagosed in early stage, most of these cases are non-small-cell lung cancer (NSCLC). The main therapeutic methods include surgery, radiotherapy, chemotherapy and targeted therapy, among which operation is the only radical method, but the therapeutic effect is disappointed.Epidermal growth factor receptor(EGFR) tyrosine kinase domain has become the fastest developing Therapeutic target now.Gefitinib and erlotinib are the most common tyrosine kinase inhibitors, and have been widely used in the treatment of patients with advanced NSCLC. The efficacy of TKIs is associated with EGFR mutation. K-ras is one of the most important members of RAS family. K-ras mutation may leading to the primary resistance of EGFR TKIs and poor prognosis of NSCLC patients. It is very necessary to comform the EGFR gene type before the treatment of patients suffering from advanced NSCLC. Currently, direct sequencing is the most common method of detection EGFR mutation, and is also as the "gold standard". But direct sequencing is complex, time-and cost-intensive, less sensitive. As a result, direct sequencing isn't able to fulfill the requirement. The Denaturing High-Performance Liquid Chromatography(DHPLC) is a new technology of high-throughput screening of gene variations.wild type and mutant DNA were detected and separated in partial denaturing condition by ion-pair-reverse-phase liquid chromatography.DHPLC is a rapid automated sensitive and specific method in mutant gene detection. TKIs had be successful in the treatment of advanced NSCLC.The neoadjuvant TKIs in operable NSCLC patients would be a new treatment model. But the heterogeneity of malignant tumors is significant. Many studies showed discrepancy in EGFR mutation between primary tumors and corresponding metastasis. We evaluated that DHPLC as a rapid detection method for EGFR mutation in NSCLC tumor specimens and compared the EGFR and K-ras genotypes between primary tumors and corresponding metastatic lymph node in NSCLC.
This research consists two parts
Part one
Detection of EGFR mutations in NSCLC tumor specimens from various ways by DHPLC and comparison of EGFR
Objective
Evaluation DHPLC as a rapid detection method for EGFR mutations of NSCLC tumor specimens from CT guided transthoracic needle biopsy of lung, lymph node biopsy and surgical resection. Correlation analysis of EGFR mutations and clinical characteristic of NSCLC patients.
Method
1.83 NSCLC patients hospitalized in guangdong general hospital from January 2008 to June 2009 were included. There were 37 tumor specimens from CT guided transthoracic needle biopsy of lung,15 from Lymph Node Biopsy, and 31 from surgical resection. The tumor specimens were divided into two, a section fixed by 10% neutral formalin and paraffin embedded was used for pathological diagnosis. The other section was put into liquid nitrogen and then transferred to -80℃low temperature refrigerator. All the patients were naive chemotherapy or targeting therapy, the specimens included in the study have be pathologically confirmed.
2. DNA extraction:The genomic DNA was extracted from lung cancer tissues.The DNA purity and content were detected by the Eppendorf nucleic acid protein cryoscope.
3. TA cloning method was used to constructed EGFR gene exon 19, exon 21 wild-type and mutant (del746-750, L858R) plasma. The 1:1,1:5,1:10,1:20,1:50,1:100 dilutions of mutant and wild type EGFR plasma DNA were using in evaluation the sensitivity of DHPLC and direct sequencing.
4. The EGFR exon 19,21 were amplified by PCR. PCR products were used for direct sequencing and DHPLC detection after being evaluated by 2% agarose gel electrophoresis.
5. The PCR products were purified.Direct sequencing of the PCR products was performed using ABI PRISM 3100 DNA Analyzer. Chromas software was used to analyze the sequencing results.
6. PCR products were detected by DHPLC without any purification. We compared the sensitivity of non-denatured condition and the partial denatured condition in the detection of the EGFR exon 19. Exon 21 was evaluated by partial denatured condition. The exon 19,21 were detected by partial denatured condition in tumor specimens.
7. Statistics analysis:All data were analyzed with SPSS 13.0 software. Chi-square test was used in examination of the relationship between EGFR mutations and gemder, smoking status, pathological type. P< 0.05 was considered statistically significant for all analyses.
Result
1. evaluation of DHPLC sensitivity:Mutant plasma DNA can be detected in the serial dilution of 1:100 by DHPLC and 1:10 by direct sequencing. The sensitivity of detecting EGFR exon 19 is similar in the condition of non-denaturing and partial denaturing.
2. DHPLC showed 22 EGFR mutations in 83 NSCLC patients,19 samples had been conformed by direct sequencing except 3 samples, the other 61 samples are wild type by DHPLC and direct sequencing. Compared with direct sequencing, the sensitivity of DHPLC was 100%, and the specificity was 95.31%.The sensitivity of detection tumor specimens from CT-guided transthoracic needle lung biopsy, lymph node Biopsy and surgical resection were 100%,and the specificity of detection tumor specimens from CT-guided transthoracic needle lung biopsy, lymph node Biopsy and surgical resection were 96.55%,100.00% and 92.00% respectively.
3. Compared with DHPLC the sensitivity of direct sequencing was 86.36%.
4. EGFR mutations were present in 37.93% female and 14.81% male(x2=5.712, P=0.017);in 32.14% adenocarcinoma and 3.70% non-adenocarcinoma(x2=8.347, P=0.004), There was no statistical difference. EGFR mutations were present in 40.74%.Feminine, non-smoking, the adenocarcinoma NSCLC patients. But the EGFR genotype was not associated with smoking status (P>0.05).
Conclusion
DHPLC is a sensitive method for screening EGFR mutations, partial denatured condition is applicable to detecting EGFR exon 19, and can detect unknown mutation. DHPLC is in accordance with the "gold standard" and is a precise rapid preliminary screening method for detection of NSCLC EGFR genotype. The tumor specimens were from CT-guided transthoracic needle lung biopsy, lymph node Biopsy and surgical resection which are the most commonly ways, the results of DHPLC is in accordance with direct sequencing. EGFR mutations were present in 22.89% of 83 NSCLC patients.Female and adenocarcinoma patients showed higher mutation frequency. Smoking status and age were no significant correlation with EGFR mutations. In conclusion DHPLC is a precise rapid preliminary screening method for detection of NSCLC EGFR genotype.
Part two Comparison of EGFR and K-ras genotypes between primary tumors and corresponding lymph node Metastases in NSCLC
Objective
Correlation analysis of EGFR and K-ras genotypes between primary tumors and corresponding metastatic lymph node in NSCLC.
Method
1. Macthed primary tumors and metastatic lymph nodes in 21 NSCLC patients hospitalized in guangdong general hospital from may 2008 to February 2010 were included. There were 14 primary tumor specimens from surgical resection and the other 7 from CT guided transthoracic needle biopsy of lung. There were 7 metastatic lymph nodes from surgical resection,11 from Mediastinoscopy and the other 3 from infraclavicula lymph node biopsy. The tumor specimens were divided into two, a section fixed by 10% neutral formalin and paraffin embedded was used for pathological diagnosis. The other section was put into liquid nitrogen and then transferred to-80℃low temperature refrigerator.
2. DNA extraction:The genomic DNA was extracted from lung cancer tissues.The DNA purity and content were detected by the Eppendorf nucleic acid protein cryoscope.
3. The EGFR exon 18-21 and K-ras codon 12,13,59 were amplified by PCR. PCR products were used for direct sequencing and DHPLC detection after being evaluated by 2% agarose gel electrophoresis.
4. The PCR products were purified.Direct sequencing of the PCR products was performed using ABI PRISM 3100 DNA Analyzer. Chromas software was used to analyze the sequencing results.
5. Statistics analysis:All data were analyzed with SPSS 13.0 software. Kappa test was used in examination of the homogeneity in EGFR and K-ras genotypes between primary tumors and corresponding metastatic lymph node in NSCLC. P< 0.05 was considered statistically significant for homogeneity. Examination of the homogeneity combined the Kappa value.
Result
A K-ras mutation was present in 4.76% of 21 patients, and was concordant in primary tumors and corresponding metastatic lymph node. We found concordant EGFR mutations in 6 patients.4 cases harbored exon 21 L858R mutation,1 case harbored exon 21 L858R and exon 20 T790M mutations,1 case harbored exon 19 delection. Discordant EGFR mutation was present in only one case, primary tumor harbored EGFR wild type, but metastatic lymph node harbored EGFR exon 19 delection. Concordant EGFR and K-ras wild type were found in 13 patients. EGFR and K-ras genotypes of primary tumors and corresponding metastatic lymph node in , NSCLC were concordant(Kappa value of EGFR and K-ras is 0.889 and 1.000, P<0.001).
Conclusion
EGFR and K-ras genotypes of primary tumors and corresponding metastatic lymph node in NSCLC were concordant by detecting fresh tissues, but we need enlarge the sample size for further research.
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