A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry.
A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry.
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DOI：
10.1371/journal.pone.0043842
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
López-Ríos F
中科院分区：
文献类型：
--
作者：
Angulo B;Conde E;Suárez-Gauthier A;Plaza C;Martínez R;Redondo P;Izquierdo E;Rubio-Viqueira B;Paz-Ares L;Hidalgo M;López-Ríos F
本研究的目的是比较两种EGFR检测方法（商业实时PCR试剂盒和特异性EGFR突变体免疫组化），直接测序，并研究两种基于PCR的方法的检测限（LOD）。我们通过直接测序分析了136例肿瘤中的21例（16%），确定了EGFR突变。有趣的是，Therascreen EGFR突变检测试剂盒能够将无法通过PCR产物直接测序分析的肿瘤表征为野生型。然后，我们比较了试剂盒的LOD和使用可用的突变肿瘤直接测序的LOD。该试剂盒能够在18个肿瘤中的9个（50%）中检测到1%稀释的总DNA中存在突变，这些肿瘤用实时定量PCR方法检测为阳性。在所有情况下，在5%稀释度下鉴定EGFR突变。当突变DNA占总DNA的30%时，测序能够在19例病例中的12例（63%）中检测到突变。使用基因定义标准品（EGFR Δ E746-A750/+和EGFR L858R/+）进行的其他实验产生了相似的结果。免疫组织化学（IHC）染色外显子19特异性抗体中看到8个9例E746-A750 del直接测序检测。两个复杂缺失的肿瘤均为阳性。在通过PCR方法检测到的5个L858R突变的肿瘤中，只有2个对外显子21特异性抗体呈阳性。两种抗体的特异性均为100%。荧光定量PCR方法的检出限低于直接测序法。突变特异性IHC产生优异的特异性。
The objective of this study is to compare two EGFR testing methodologies (a commercial real-time PCR kit and a specific EGFR mutant immunohistochemistry), with direct sequencing and to investigate the limit of detection (LOD) of both PCR-based methods. We identified EGFR mutations in 21 (16%) of the 136 tumours analyzed by direct sequencing. Interestingly, the Therascreen EGFR Mutation Test kit was able to characterize as wild-type one tumour that could not be analyzed by direct sequencing of the PCR product. We then compared the LOD of the kit and that of direct sequencing using the available mutant tumours. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in nine of the 18 tumours (50%), which tested positive with the real-time quantitative PCR method. In all cases, EGFR mutation was identified at a dilution of 5%. Where the mutant DNA represented 30% of the total DNA, sequencing was able to detect mutations in 12 out of 19 cases (63%). Additional experiments with genetically defined standards (EGFR ΔE746-A750/+ and EGFR L858R/+) yielded similar results. Immunohistochemistry (IHC) staining with exon 19-specific antibody was seen in eight out of nine cases with E746-A750del detected by direct sequencing. Neither of the two tumours with complex deletions were positive. Of the five L858R-mutated tumours detected by the PCR methods, only two were positive for the exon 21-specific antibody. The specificity was 100% for both antibodies. The LOD of the real-time PCR method was lower than that of direct sequencing. The mutation specific IHC produced excellent specificity.
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