Section of Cancer Genomics, Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Genes Chromosomes Cancer. 2012 May;51(5):490-500. doi: 10.1002/gcc.21937. Epub 2012 Feb 15.
To identify the genetic drivers of colorectal tumorigenesis, we applied array comparative genomic hybridization (aCGH) to 13 formalin-fixed paraffin-embedded (FFPE) samples of early, localized human colon adenocarcinomas arising in high-grade adenomas (so-called "malignant polyps"). These lesions are small and hence the amount of DNA is limited. Additionally, the quality of DNA is compromised due to the fragmentation as a consequence of formalin fixation. To overcome these problems, we optimized a newly developed isothermal whole genome amplification system (NuGEN Ovation® WGA FFPE System). Starting with 100 ng of FFPE DNA, the amplification system produced 4.01 ± 0.29 μg (mean ± standard deviation) of DNA. The excellent quality of amplified DNA was further indicated by a high signal-to-noise ratio and a low derivative log(2) ratio spread. Both, the amount of amplified DNA and aCGH performance were independent of the age of the FFPE blocks and the associated degradation of the extracted DNA. We observed losses of chromosome arms 5q and 18q in the adenoma components of the malignant polyp samples, while the embedded early carcinomas revealed losses of 8p, 17p, and 18, and gains of 7, 13, and 20. Aberrations detected in the adenoma components were invariably maintained in the embedded carcinomas. This approach demonstrates that using isothermally whole genome amplified FFPE DNA is technically suitable for aCGH. In addition to demonstrating the clonal origin of the adenoma and carcinoma part within a malignant polyp, the gain of chromosome arm 20q was an indicator for progression from adenoma to carcinoma.
为了鉴定结直肠肿瘤形成的遗传驱动因素,我们应用比较基因组杂交(array comparative genomic hybridization,aCGH)对 13 例福尔马林固定石蜡包埋(formalin-fixed paraffin-embedded,FFPE)的早发局限性人结肠腺癌样本进行分析,这些肿瘤来源于高级别腺瘤(所谓的“恶性息肉”)。这些病变较小,因此 DNA 量有限。此外,由于福尔马林固定导致的片段化,DNA 的质量也受到影响。为了克服这些问题,我们优化了一种新开发的等温全基因组扩增系统(NuGEN Ovation® WGA FFPE System)。从 100ng FFPE DNA 开始,扩增系统产生了 4.01 ± 0.29μg(平均值 ± 标准差)的 DNA。扩增 DNA 的高质量还进一步通过高信号与噪声比和低衍生 log(2)比值扩展来表明。扩增 DNA 的量和 aCGH 性能均不受 FFPE 块的年龄以及相关提取 DNA 降解的影响。我们观察到恶性息肉样本中腺瘤成分的 5q 和 18q 染色体臂缺失,而嵌入的早期癌显示 8p、17p 和 18 的缺失,7、13 和 20 的获得。在腺瘤成分中检测到的异常情况在嵌入的癌中始终保持不变。这种方法表明,使用等温全基因组扩增 FFPE DNA 技术上适合 aCGH。除了证明恶性息肉中腺瘤和癌部分的克隆起源外,染色体臂 20q 的获得是从腺瘤进展为癌的指标。