应用 COLD-PCR 技术对非小细胞肺癌支气管内超声引导经支气管针吸活检标本进行 EGFR 和 KRAS 基因突变检测。
Screening for EGFR and KRAS mutations in endobronchial ultrasound derived transbronchial needle aspirates in non-small cell lung cancer using COLD-PCR.
机构信息
Division of Asthma, Allergy & Lung Biology, King's College London, London, United Kingdom.
出版信息
PLoS One. 2011;6(9):e25191. doi: 10.1371/journal.pone.0025191. Epub 2011 Sep 19.
EGFR mutations correlate with improved clinical outcome whereas KRAS mutations are associated with lack of response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Endobronchial ultrasound (EBUS)-transbronchial needle aspiration (TBNA) is being increasingly used in the management of NSCLC. Co-amplification at lower denaturation temperature (COLD)-polymerase chain reaction (PCR) (COLD-PCR) is a sensitive assay for the detection of genetic mutations in solid tumours. This study assessed the feasibility of using COLD-PCR to screen for EGFR and KRAS mutations in cytology samples obtained by EBUS-TBNA in routine clinical practice. Samples obtained from NSCLC patients undergoing EBUS-TBNA were evaluated according to our standard clinical protocols. DNA extracted from these samples was subjected to COLD-PCR to amplify exons 18-21 of EGFR and exons two and three of KRAS followed by direct sequencing. Mutation analysis was performed in 131 of 132 (99.3%) NSCLC patients (70F/62M) with confirmed lymph node metastases (94/132 (71.2%) adenocarcinoma; 17/132 (12.8%) squamous cell; 2/132 (0.15%) large cell neuroendocrine; 1/132 (0.07%) large cell carcinoma; 18/132 (13.6%) NSCL-not otherwise specified (NOS)). Molecular analysis of all EGFR and KRAS target sequences was achieved in 126 of 132 (95.5%) and 130 of 132 (98.4%) of cases respectively. EGFR mutations were identified in 13 (10.5%) of fully evaluated cases (11 in adenocarcinoma and two in NSCLC-NOS) including two novel mutations. KRAS mutations were identified in 23 (17.5%) of fully analysed patient samples (18 adenocarcinoma and five NSCLC-NOS). We conclude that EBUS-TBNA of lymph nodes infiltrated by NSCLC can provide sufficient tumour material for EGFR and KRAS mutation analysis in most patients, and that COLD-PCR and sequencing is a robust screening assay for EGFR and KRAS mutation analysis in this clinical context.
表皮生长因子受体（EGFR）突变与非小细胞肺癌（NSCLC）患者的临床获益相关，而 KRAS 突变则与酪氨酸激酶抑制剂治疗无反应相关。支气管内超声（EBUS）-经支气管针吸活检术（TBNA）在 NSCLC 的治疗中应用越来越广泛。在实体瘤中，低变性温度共扩增（COLD）-聚合酶链反应（PCR）（COLD-PCR）是一种用于检测基因突变的敏感检测方法。本研究评估了在常规临床实践中使用 EBUS-TBNA 获得的细胞学样本中使用 COLD-PCR 筛查 EGFR 和 KRAS 突变的可行性。根据我们的标准临床方案评估了来自接受 EBUS-TBNA 的 NSCLC 患者的样本。从这些样本中提取的 DNA 用于 COLD-PCR 扩增 EGFR 的外显子 18-21 和 KRAS 的外显子 2 和 3，然后直接测序。对 132 例（99.3%）经证实有淋巴结转移的 NSCLC 患者（70 例女性/62 例男性）中的 131 例（99.3%）进行了突变分析（94 例（71.2%）腺癌；17 例（12.8%）鳞状细胞癌；2 例（0.15%）大细胞神经内分泌癌；1 例（0.07%）大细胞癌；18 例（13.6%）NSCL-未特指）。对所有 EGFR 和 KRAS 靶序列的分子分析在 132 例患者中的 126 例（95.5%）和 130 例（98.4%）中获得。在 13 例（10.5%）充分评估的病例中（11 例腺癌和 2 例 NSCLC-NOS）发现了 EGFR 突变，其中包括 2 种新突变。在 23 例（17.5%）充分分析的患者样本中发现了 KRAS 突变（18 例腺癌和 5 例 NSCLC-NOS）。我们得出结论，EBUS-TBNA 对受 NSCLC 浸润的淋巴结进行穿刺可以为大多数患者提供足够的肿瘤材料，用于 EGFR 和 KRAS 突变分析，并且 COLD-PCR 和测序是该临床背景下 EGFR 和 KRAS 突变分析的可靠筛选检测方法。
Zotero 插件