Park Sojung, Hur Jae Young, Lee Kye Young, Lee Jae Cheol, Rho Jin Kyung, Shin Sun Hwa, Choi Chang-Min
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Clin Chem Lab Med. 2017 Aug 28;55(10):1489-1495. doi: 10.1515/cclm-2016-0302.
Much attention has been focused on epidermal growth factor receptor (EGFR) mutation testing since the introduction of EGFR-tyrosine kinase inhibitors have improved survival in EGFR-positive lung cancer patients. Liquid biopsy using circulating tumor cells or cell-free DNA (cfDNA) has enabled less invasive testing, but requires a highly sensitive method. To date, liquid biopsy using bronchoalveolar lavage (BAL) fluid has rarely been used.
From 20 patients with lung adenocarcinoma, we isolated cfDNA from 20 samples of cell-free BAL fluid and 19 cell-free bronchial washing samples. cfDNA was examined for EGFR mutations using peptide nucleic acid (PNA)-mediated PCR clamping method. In cases where the results from the tumor biopsy and BAL-derived cfDNA test were not consistent, PANAMutyper™ R EGFR kit was used along with PNA clamping-assisted fluorescence melting curve analysis.
We included 17 patients with advanced stage disease and three with non-advanced stage disease. Tumor biopsy detected EGFR mutations in 12 of the patients. One patient had a p.L858R mutation and a de novo p.T790M mutation. The results from PNA-mediated PCR clamping were 75.0% (9/12) concordant with the tumor biopsy results for EGFR mutation status. PANAMutyper with fluorescence melting curve analysis was performed in three cases, which detected EGFR mutations in two more patients (11/12, 91.7%). EGFR mutations were detected in the cfDNA extracted from two bronchial washing samples.
cfDNA from BAL fluid could be used for molecular testing of EGFR mutations and identification of p.T790M mutations, with an easily applicable method.
自表皮生长因子受体（EGFR）酪氨酸激酶抑制剂提高了EGFR阳性肺癌患者的生存率以来，EGFR突变检测受到了广泛关注。使用循环肿瘤细胞或游离DNA（cfDNA）进行液体活检实现了侵入性较小的检测，但需要高度灵敏的方法。迄今为止，使用支气管肺泡灌洗（BAL）液进行液体活检很少被采用。
从20例肺腺癌患者中，我们从20份游离BAL液样本和19份游离支气管冲洗样本中分离出cfDNA。使用肽核酸（PNA）介导的PCR钳夹法检测cfDNA中的EGFR突变。在肿瘤活检结果与BAL衍生的cfDNA检测结果不一致的情况下，使用PANAMutyper™ R EGFR试剂盒并结合PNA钳夹辅助荧光熔解曲线分析。
我们纳入了17例晚期疾病患者和3例非晚期疾病患者。肿瘤活检在12例患者中检测到EGFR突变。1例患者有p.L858R突变和新发p.T790M突变。PNA介导的PCR钳夹结果与肿瘤活检的EGFR突变状态结果的一致性为75.0%（9/12）。对3例患者进行了带有荧光熔解曲线分析的PANAMutyper检测，又检测到另外2例患者有EGFR突变（11/12，91.7%）。在从两份支气管冲洗样本中提取的cfDNA中检测到了EGFR突变。
BAL液中的cfDNA可用于EGFR突变的分子检测和p.T790M突变的鉴定，且方法易于应用。
