MET, HGF, EGFR, and PXN gene copy number in lung cancer using DNA extracts from FFPE archival samples and prognostic significance.
MET, HGF, EGFR, and PXN gene copy number in lung cancer using DNA extracts from FFPE archival samples and prognostic significance.
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DOI：
10.1615/jenvironpatholtoxicoloncol.v28.i2.10
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Salgia R
中科院分区：
文献类型：
--
作者：
Kanteti R;Yala S;Ferguson MK;Salgia R
对MET、肝细胞生长因子（HGF， MET配体）、表皮生长因子受体（EGFR）和帕西林（PXN）等肺肿瘤发生过程中一些重要分子的基因拷贝数分析可能决定治疗类型和预后。福尔马林固定石蜡包埋（FFPE）档案肿瘤组织样本是确定OncoGenome中关键分子变化的极好来源，然而现有的提取方法产生的基因组DNA片段质量相对较差。虽然FISH是确定基因扩增的首选方法，但当有相当高质量的基因组DNA时，可以使用更快速的qPCR技术来确定基因拷贝数。我们在此报告了一种基于微波/chelex-100处理的相对快速的方法，该方法产生的基因组DNA片段范围从1到12 Kb甚至更大，从而证明了其优越的质量。β-珠蛋白基因的基因组PCR结果可靠，重复性好。提取DNA的步骤保持在最低限度，而不是沉淀DNA，我们保存了基因组DNA提取物，以防止DNA产量的损失。我们发现提取物是稳定的，适合qPCR和突变分析。通过肺腺癌FFPE样本和来源于肺腺癌的细胞系，我们证明了肺腺癌组织样本中MET基因拷贝数比EGFR、HGF和PXN优先增加，并且与更好的预后呈正相关。相比之下，从25个非小细胞肺癌细胞系中提取的基因组DNA对所有四个评估基因的基因拷贝数相对较高。我们的结果表明，基于微波/chelex-100的方法可以获得高质量的基因组DNA提取物，可用于复杂DNA分析，如基因拷贝数的测定。此外，我们的数据表明，腺癌细胞系可能在离体条件下进化，因此在遗传学研究中，有必要使用原发肿瘤来得出关于肺肿瘤的一般性结论。
Gene copy number analysis for some of the important molecules in lung tumorogenesis such as MET, hepatocyte growth factor (HGF, ligand for MET), epidermal growth factor receptor (EGFR) and paxillin (PXN) is likely to determine both the type of treatment and prognosis. Formalin-fixed paraffin-embedded (FFPE) archival tumor tissue samples are an excellent source for determining key molecular changes in the OncoGenome, however existing extraction procedures yield relatively poor quality genomic DNA fragments. Although FISH is the method of choice for determining amplification of a gene, a more rapid qPCR technique to determine gene copy number can be used when reasonably good quality genomic DNA is available. We report here a relatively rapid method based on microwave/chelex-100 treatment that gives rise to genomic DNA fragments ranging from 1 to 12 Kb and beyond, thereby attesting to its superior quality. Genomic PCR for β-globin gene gave reliable and reproducible results. The number of steps for extracting the DNA was kept to a minimum, and instead of precipitating the DNA, we preserved the genomic DNA extracts so as to prevent a loss in DNA yield. We found the extracts to be stable and amenable to qPCR and mutational analysis. Using lung adenocarcinoma FFPE samples and cell lines derived from lung adenocarcinomas, we demonstrated that the gene copy number for MET in lung adenocarcinoma tissue samples was preferentially increased over EGFR, HGF and PXN and that it positively correlated with better prognosis. In contrast, the genomic DNA extracted from twenty five NSCLC cell lines gave relatively higher gene copy number for all the four genes evaluated. Our results indicate that the microwave/chelex-100 based method yields good quality genomic DNA extracts that can be used for complex DNA analysis such as determination of gene copy number. In addition, our data demonstrated that the adenocarcinoma cell lines potentially evolved under ex vivo conditions, and therefore in genetic studies it is imperative to use primary tumors for generalized conclusions about lung tumors.
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影响因子：
2.7
作者：
WALSH, PS;METZGER, DA;HIGUCHI, R
通讯作者：
HIGUCHI, R
影响因子：
3.4
作者：
SEPP, R;SZABO, I;SAKAMOTO, H
通讯作者：
SAKAMOTO, H
DOI：
10.1073/pnas.0710370104
发表时间：
2007-12-26
影响因子：
11.1
作者：
Bean, James;Brennan, Cameron;Pao, William
通讯作者：
Pao, William
影响因子：
3.7
作者：
Ma, Patrick C.;Tretiakova, Maria S.;MacKinnon, Alexander C.;Ramnath, Nithya;Johnson, Candace;Dietrich, Sascha;Seiwert, Tanguy;Christensen, James G.;Jagadeeswaran, Ramasamy;Krausz, Thomas;Vokes, Everett E.;Husain, Aliya N.;Salgia, Ravi
通讯作者：
Salgia, Ravi
影响因子：
11.2
作者：
Jagadeeswaran, Ramasamy;Surawska, Hanna;Salgia, Ravi
通讯作者：
Salgia, Ravi