Quantifying EGFR Alterations in the Lung Cancer Genome with Nanofluidic Digital PCR Arrays
Quantifying EGFR Alterations in the Lung Cancer Genome with Nanofluidic Digital PCR Arrays
复制标题
DOI：
10.1373/clinchem.2009.134973
复制
发表时间：
2010-04-01
影响因子：
9.3
通讯作者：
Ma, Patrick C.
中科院分区：
文献类型：
--
作者：
Wang, Jun;Ramakrishnan, Ramesh;Ma, Patrick C.
背景：EGFR[表皮生长因子受体（红细胞白血病病毒（v- erbb -b）癌基因同源物，鸟类）]基因已知在晚期肺癌中包含基因扩增和激酶突变的基因组改变，这些基因扩增和激酶突变可预测EGFR靶向抑制剂的临床反应。在肺癌肿瘤中检测这种分子变化的方法是需要的。方法：我们使用纳米流体数字PCR阵列平台和16个细胞系和20个来自切除肿瘤（I-III期）的基因组DNA样本来量化EGFR基因的相对拷贝数，并检测肺癌中突变的EGFR等位基因。我们通过计算每个面板中EGFR分子数（用6-羧基荧光素标记的Scorpion （TM）测定）与单拷贝基因RPP30（核糖核酸酶P/MRP 30kDa亚基）分子数（用6-羧基- x -罗丹明标记的TaqMan T测定）的比例来评估EGFR基因拷贝的相对数量。为了检测EGFR L858R（21外显子）突变和帧内19外显子缺失，我们在DxS/Qiagen EGFR29突变检测试剂盒中使用ARMS （TM）和Scorpion技术用于数字PCR阵列。结果：数字阵列检测并量化了福尔马林固定、石蜡包埋的早期可切除肺肿瘤样本中罕见的吉非替尼/厄洛替尼致敏EGFR突变（丰度为0.02%-9.26%），且基因拷贝数未增加。我们的研究结果还表明，在这些早期肺肿瘤中，临床相关的EGFR突变等位基因存在肿瘤内分子异质性。结论：数字PCR阵列平台可以在单分子水平上对癌基因（如EGFR）进行表征和定量。使用这种纳米流体平台可以更深入地了解临床相关激酶突变在肺肿瘤进展不同阶段的具体作用，并可能有助于预测对egfr靶向抑制剂的临床反应。(C) 2010年美国临床化学学会
BACKGROUND: The EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] gene is known to harbor genomic alterations in advanced lung cancer involving gene amplification and kinase mutations that predict the clinical response to EGFR-targeted inhibitors. Methods for detecting such molecular changes in lung cancer tumors are desirable.METHODS: We used a nanofluidic digital PCR array platform and 16 cell lines and 20 samples of genomic DNA from resected tumors (stages I-III) to quantify the relative numbers of copies of the EGFR gene and to detect mutated EGFR alleles in lung cancer. We assessed the relative number of EGFR gene copies by calculating the ratio of the number of EGFR molecules (measured with a 6-carboxyfluorescein-labeled Scorpion (TM) assay) to the number of molecules of the single-copy gene RPP30 (ribonuclease P/MRP 30kDa subunit) (measured with a 6-carboxy-X-rhodamine labeled TaqMan T assay) in each panel. To assay for the EGFR L858R (exon 21) mutation and exon 19 in-frame deletions, we used the ARMS (TM) and Scorpion technologies in a DxS/Qiagen EGFR29 Mutation Test Kit for the digital PCR array.RESULTS: The digital array detected and quantified rare gefitinib/erlotinib-sensitizing EGFR mutations (0.02%-9.26% abundance) that were present in formalin-fixed, paraffin-embedded samples of early-stage resectable lung tumors without an associated increase in gene copy number. Our results also demonstrated the presence of intratumor molecular heterogeneity for the clinically relevant EGFR mutated alleles in these early-stage lung tumors.CONCLUSIONS: The digital PCR array platform allows characterization and quantification of oncogenes, such as EGFR, at the single-molecule level. Use of this nanofluidics platform may provide deeper insight into the specific roles of clinically relevant kinase mutations during different stages of lung tumor progression and may be useful in predicting the clinical response to EGFR-targeted inhibitors. (C) 2010 American Association for Clinical Chemistry