Chen Yi-Lin, Lee Chung-Ta, Lu Cheng-Chan, Yang Shu-Ching, Chen Wan-Li, Lee Yang-Cheng, Yang Chung-Hsien, Peng Shu-Ling, Su Wu-Chou, Chow Nan-Haw, Ho Chung-Liang
Molecular Diagnostics Laboratory, Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan.
Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University Hospital, Tainan, Taiwan.
PLoS One. 2016 Jun 28;11(6):e0158125. doi: 10.1371/journal.pone.0158125. eCollection 2016.
Analyzing EGFR mutations and detecting ALK gene fusion are indispensable when planning to treat pulmonary adenocarcinoma. Malignant pleural effusion (MPE) is a devastating complication of lung cancer and sometimes the only source for mutation analysis. The percentage of tumor cells in the pleural effusion may be low; therefore, mutant enrichment is required for a successful analysis. The EGFR mutation status in MPE was determined using three methods: (1) PCR sequencing of genomic DNA (direct sequencing), (2) mutant-enriched PCR sequencing of genomic DNA using peptide nucleic acid (PNA-sequencing), and (3) PCR sequencing of cDNA after reverse transcription for cellular RNA (RNA-sequencing). RT-PCR was also used to test cases for ALK gene fusion. PNA-sequencing and RNA-sequencing had similar analytical sensitivities (< 1%), which indicates similar enrichment capabilities. The clinical sensitivity in 133 cases when detecting the common EGFR exon 19 and exon 21 mutations was 56.4% (75/133) for direct sequencing, 63.2% (84/133) for PNA-sequencing, and 65.4% (87/133) for RNA-sequencing. RT-PCR and sequencing showed 5 cases (3.8%) with ALK gene fusion. All had wild-type EGFR. For EGFR analysis of MPE, RNA-sequencing is at least as sensitive as PNA-sequencing but not limited to specific mutations. Detecting ALK fusion can be incorporated in the same RNA workflow. Therefore, RNA is a better source for comprehensive molecular diagnoses in MPE.
在计划治疗肺腺癌时，分析表皮生长因子受体（EGFR）突变和检测间变性淋巴瘤激酶（ALK）基因融合是必不可少的。恶性胸腔积液（MPE）是肺癌的一种严重并发症，有时是突变分析的唯一来源。胸腔积液中的肿瘤细胞百分比可能较低；因此，为了成功进行分析，需要进行突变富集。使用三种方法确定MPE中的EGFR突变状态：（1）基因组DNA的聚合酶链反应（PCR）测序（直接测序），（2）使用肽核酸对基因组DNA进行突变富集PCR测序（肽核酸测序），以及（3）细胞RNA逆转录后cDNA的PCR测序（RNA测序）。逆转录聚合酶链反应（RT-PCR）也用于检测ALK基因融合的病例。肽核酸测序和RNA测序具有相似的分析灵敏度（<1%），这表明它们具有相似的富集能力。在检测常见的EGFR外显子19和外显子21突变时，133例患者中直接测序的临床灵敏度为56.4%（75/133），肽核酸测序为63.2%（84/133），RNA测序为65.4%（87/133）。RT-PCR和测序显示5例（3.8%）存在ALK基因融合。所有病例的EGFR均为野生型。对于MPE的EGFR分析，RNA测序至少与肽核酸测序一样灵敏，但不限于特定突变。检测ALK融合可纳入相同的RNA工作流程。因此，RNA是MPE综合分子诊断的更好来源。
