腺癌患者纤维支气管镜活检标本的表皮生长因子受体基因检测
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摘要
目的：
通过蝎形探针扩增阻滞突变系统和免疫组化法,测定腺癌患者支气管镜黏膜活检标本中EGFR基因突变的检出率以及两者之间一致性,分析小标本EGFR基因突变检测的影响因素,并进一步探讨腺癌患者小标本EGFR基因检测作为早期诊疗参考指标在临床应用中的意义。
方法：
回顾2009年1月—2011年11月于北京协和医院行支气管镜黏膜活检确诊腺癌患者的小活检标本108例,收集所有患者的相关临床资料,镜检HE切片肿瘤细胞比例和数量,应用蝎形探针扩增阻滞突变系统检测小活检标本EGFR突变,应用EGFR E746-A750del单抗和21L858R单抗检测小活检标本EGFR常见突变的蛋白表达。
结果：
108例患者中,92例患者除去临床诊断所需标本后有标本剩余可以进行基因突变检测,检出率为85.2%。男性、老年、Ⅳ期、支气管镜下表现为增生型、取材小于等于4次、中分化肺癌、肿瘤细胞数量小于200个/高倍视野的患者无标本剩余的概率更高,但均无统计学意义(P<0.05)。应用Scorpions-ARMS法,92例标本DNA都可以检测到扩增曲线,检测成功率为100%。33例肺腺癌患者存在EGFR基因突变,突变率为35.8%。其中15例(16.3%)存在19号外显子缺失突变(19del),7例(18.5%)存在21号外显子点突变(包括15例21L858R突变和2例21L861Q突变),1例(3.0%)存在20号外显子插入突变,1例(3.0%)存在20号外显子点突变(20S768I),1例(3.0%)同时存在19del和21L858R突变。女性、无吸烟史组突变率更高,但结果无统计学意义(P>0.05),女性较男性发生21L858R突变的概率更高(P＝0.022),支气管镜下为浸润型表现组EGFR突变率更高(P=0.043)。年龄、临床分期、活检部位、活检取样次数、是否明确为腺癌、肿瘤分化程度、肿瘤细胞数量、肿瘤细胞比例、免疫组化使用切片数量与EGFR突变无明显相关(P>0.05)。基因突变组0S明显长于野生组总生存期(P=0.036)。E746-A750的阳性表达率为17.8%(16/90),与Scorpions ARMS法相比,敏感性为73.3%,特异性为93.3%,一致性一般(kappa=0.649); L858R的阳性表达率为21.4%(19／89),与Scorpions ARMS法相比,敏感性为93.3%,特异性为93.2%,一致性较好(kappa=0.783)。
结论：
腺癌患者支气管镜活检标本EGFR基因突变的检出率为85.2%。取材少可能导致标本过少,无法进行EGFR基因检测。EGFR基因突变与性别、支气管镜下表现、总生存期有关,与肿瘤分化程度、临床分期无关。使用突变特异的抗体进行免疫组化染色可以进行EGFR突变筛查。
ObjectiveTo explore the feasibility of performing epidermal growth factor receptor mutation analysis on low-volume transbronchial biopsy samples byscorpions amplification refractory mutation system and by immunohistochemistry, and compare the sensitivity of these methods.
Methods108patients with transbronchial biopsy tumor tissue samples and histologically confirmed adenocarcinoma, from January2009through December2011at Peking Union Medical College Hospital were enrolled. Clinical mannifestations were reviewed. All samples were microscopically examined for tumor cell ratio. EGFR mutation was assessed from DNA of the paraffin-embedded specimens by Scorpions ARMS, immunohistochemistry staining was performed using EGFR E746-A750del and21L858Rmutant antibody.
ResultsIn108patients,92patientshad adequatespecimens left for analyses, and the detection rate was85.2%for low-volume biospsy samples.16patients had specimens with inadequate DNA for analyses, and were more frequently in male, the elder, samples with few tumor cells and biopsy less than4times and so on. Scorpions ARMS method were successfully performed in all92cases, and33EGFR mutations(35.8%) were detectedfrom DNA extracted from transbronchial biopsy tumor tissue samples, including16exon19in-frame deletion,17exon21point mutation(21L858R and21L861Q),1exon20insertion,1exon20point mutation(20S768I) and1with both19del and21L858Rmutations. The frequency of EGFR mutations was higher in female and non-smokers.21L858R mutation was more frequently in female than man(P=0.022). Infiltrating pattern under transbroncoscopyshowed higer mutation ratecompared with other patterns(P=0.043). Patients with EGFR mutations had longer overall survival than wildtype(P=0.036).16cases (17.8%)were scored positive for the exon19EGFR E746-A750mutant antibody, showed a sensitivity of73.3%and a specificity of93.3%compared with Scorpions ARMS, andthe concordance was moderate(kappa=0.649) between the two methods.19cases (21.4%) were scored positive for the exon21EGFR L858R mutant antibody, showed a sensitivity of93.3%and a specificity of93.2%compared with Scorpions ARMS, and the concordance was good (kappa=0.783).
ConclusionEpidermal growth factor receptor mutation analysis can be performed on85.2%low-volume transbronchial biopsy samples by scorpions amplification refractory mutation system and by immunohistochemistry. Patients havefew biopsy times may have inadequate DNA for analyses. EGFR mutation status has relation with gender, tumor manifestations under transbronscopy, overall survival, but not with tumor differentiation and clinical stage. Immunohistochemistry with EGFR mutant-specific antibodies could be used as a screen to identify most mutations from low-volume biopsy samples.
通过蝎形探针扩增阻滞突变系统和免疫组化法,测定腺癌患者支气管镜黏膜活检标本中EGFR基因突变的检出率以及两者之间一致性,分析小标本EGFR基因突变检测的影响因素,并进一步探讨腺癌患者小标本EGFR基因检测作为早期诊疗参考指标在临床应用中的意义。
方法：
回顾2009年1月—2011年11月于北京协和医院行支气管镜黏膜活检确诊腺癌患者的小活检标本108例,收集所有患者的相关临床资料,镜检HE切片肿瘤细胞比例和数量,应用蝎形探针扩增阻滞突变系统检测小活检标本EGFR突变,应用EGFR E746-A750del单抗和21L858R单抗检测小活检标本EGFR常见突变的蛋白表达。
结果：
108例患者中,92例患者除去临床诊断所需标本后有标本剩余可以进行基因突变检测,检出率为85.2%。男性、老年、Ⅳ期、支气管镜下表现为增生型、取材小于等于4次、中分化肺癌、肿瘤细胞数量小于200个/高倍视野的患者无标本剩余的概率更高,但均无统计学意义(P<0.05)。应用Scorpions-ARMS法,92例标本DNA都可以检测到扩增曲线,检测成功率为100%。33例肺腺癌患者存在EGFR基因突变,突变率为35.8%。其中15例(16.3%)存在19号外显子缺失突变(19del),7例(18.5%)存在21号外显子点突变(包括15例21L858R突变和2例21L861Q突变),1例(3.0%)存在20号外显子插入突变,1例(3.0%)存在20号外显子点突变(20S768I),1例(3.0%)同时存在19del和21L858R突变。女性、无吸烟史组突变率更高,但结果无统计学意义(P>0.05),女性较男性发生21L858R突变的概率更高(P＝0.022),支气管镜下为浸润型表现组EGFR突变率更高(P=0.043)。年龄、临床分期、活检部位、活检取样次数、是否明确为腺癌、肿瘤分化程度、肿瘤细胞数量、肿瘤细胞比例、免疫组化使用切片数量与EGFR突变无明显相关(P>0.05)。基因突变组0S明显长于野生组总生存期(P=0.036)。E746-A750的阳性表达率为17.8%(16/90),与Scorpions ARMS法相比,敏感性为73.3%,特异性为93.3%,一致性一般(kappa=0.649); L858R的阳性表达率为21.4%(19／89),与Scorpions ARMS法相比,敏感性为93.3%,特异性为93.2%,一致性较好(kappa=0.783)。
结论：
腺癌患者支气管镜活检标本EGFR基因突变的检出率为85.2%。取材少可能导致标本过少,无法进行EGFR基因检测。EGFR基因突变与性别、支气管镜下表现、总生存期有关,与肿瘤分化程度、临床分期无关。使用突变特异的抗体进行免疫组化染色可以进行EGFR突变筛查。
ObjectiveTo explore the feasibility of performing epidermal growth factor receptor mutation analysis on low-volume transbronchial biopsy samples byscorpions amplification refractory mutation system and by immunohistochemistry, and compare the sensitivity of these methods.
Methods108patients with transbronchial biopsy tumor tissue samples and histologically confirmed adenocarcinoma, from January2009through December2011at Peking Union Medical College Hospital were enrolled. Clinical mannifestations were reviewed. All samples were microscopically examined for tumor cell ratio. EGFR mutation was assessed from DNA of the paraffin-embedded specimens by Scorpions ARMS, immunohistochemistry staining was performed using EGFR E746-A750del and21L858Rmutant antibody.
ResultsIn108patients,92patientshad adequatespecimens left for analyses, and the detection rate was85.2%for low-volume biospsy samples.16patients had specimens with inadequate DNA for analyses, and were more frequently in male, the elder, samples with few tumor cells and biopsy less than4times and so on. Scorpions ARMS method were successfully performed in all92cases, and33EGFR mutations(35.8%) were detectedfrom DNA extracted from transbronchial biopsy tumor tissue samples, including16exon19in-frame deletion,17exon21point mutation(21L858R and21L861Q),1exon20insertion,1exon20point mutation(20S768I) and1with both19del and21L858Rmutations. The frequency of EGFR mutations was higher in female and non-smokers.21L858R mutation was more frequently in female than man(P=0.022). Infiltrating pattern under transbroncoscopyshowed higer mutation ratecompared with other patterns(P=0.043). Patients with EGFR mutations had longer overall survival than wildtype(P=0.036).16cases (17.8%)were scored positive for the exon19EGFR E746-A750mutant antibody, showed a sensitivity of73.3%and a specificity of93.3%compared with Scorpions ARMS, andthe concordance was moderate(kappa=0.649) between the two methods.19cases (21.4%) were scored positive for the exon21EGFR L858R mutant antibody, showed a sensitivity of93.3%and a specificity of93.2%compared with Scorpions ARMS, and the concordance was good (kappa=0.783).
ConclusionEpidermal growth factor receptor mutation analysis can be performed on85.2%low-volume transbronchial biopsy samples by scorpions amplification refractory mutation system and by immunohistochemistry. Patients havefew biopsy times may have inadequate DNA for analyses. EGFR mutation status has relation with gender, tumor manifestations under transbronscopy, overall survival, but not with tumor differentiation and clinical stage. Immunohistochemistry with EGFR mutant-specific antibodies could be used as a screen to identify most mutations from low-volume biopsy samples.
引文
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