Verification of Wild-Type EGFR Status in Non-Small Cell Lung Carcinomas Using a Mutant-Enriched PCR on Selected Cases
Verification of Wild-Type EGFR Status in Non-Small Cell Lung Carcinomas Using a Mutant-Enriched PCR on Selected Cases
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DOI：
10.1016/j.jmoldx.2014.05.007
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发表时间：
2014-09-01
影响因子：
4.1
通讯作者：
Ho, Chung-Liang
中科院分区：
文献类型：
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作者：
Chen, Yi-Lin;Lu, Cheng-Chan;Ho, Chung-Liang
肺腺癌的靶向治疗需要进行表皮生长因子受体（EGFR）基因分型。由于假阴性结果可能使患者无法接受适当的靶向治疗，因此对EGFR基因分型的不确定结果进行复查是可取的。对346例肺癌患者使用一种商业试剂盒检测EGFR突变；其中9例在后期循环中出现上升的实时扩增曲线。还使用肽核酸锁核酸富集突变的聚合酶链反应（PNA测序）对这些病例进行了研究。9例不确定病例中有6例存在EGFR突变。这些病例可能在商业试剂盒的检测限附近含有少量突变DNA。使用PNA测序对20例明确的野生型病例进行了再次确认。我们注意到美国病理学家协会的一份能力验证材料显示出可疑的上升曲线，并最终证实在第20外显子存在H773_V774insPH突变，而商业试剂盒中未针对此设计特异性引物。使用克隆的DNA片段进一步研究表明，上升曲线很可能是由相似但不完全相同的序列之间的交叉反应导致的。尽可能降低假阴性结果的数量是可取的，但对所有野生型病例进行复查是不切实际的。我们观察到的后期上升曲线有助于识别可疑病例以便进行复查。建议使用第二种方法，如PNA测序，来验证野生型病例。
EGFR genotyping is required for targeted therapy of lung adenocarcinoma. Because a false-negative result might prevent a patient from receiving appropriate targeted therapies, it is desirable to recheck equivocal results of EGFR genotyping. A cohort of 346 Lung cancers was tested with a commercial kit for EGFR mutations; nine of the cases had upward real-time amplification curves at late cycles. They were also investigated using mutant-enriched PCR with peptide nucleic acid locked nucleic acid (PNA-sequencing). Six of the nine equivocal cases harbored EGFR mutations. These cases likely had a small amount of mutant DNA near the detection limit of the commercial kit. Twenty nonequivocal, wild-type cases were reconfirmed using PNA-sequencing. We noticed a College of American Pathologists proficiency test material that showed a suspicious upward curve and eventually proved to have an H773_V774insPH in exon 20, for which a specific primer was not designed in the commercial kit. Further study using cloned DNA fragments showed that the upward curve most likely resulted from cross-reaction between similar, but nonidentical, sequences. It is desirable to keep the number of false-negative results as Low as possible, but rechecking all wild-type cases is impractical. The late upward curves we observed helped identify suspicious cases for rechecking. A second method, such as PNA-sequencing, is recommended to verify wild-type cases.