Cathay General Hospital, College of Medicine, Fu-Jen Catholic University, Taipei, Taiwan.
Pathology. 2012 Jan;44(1):33-41. doi: 10.1097/PAT.0b013e32834e411e.
In the study of tumour genetics, formalin fixed, paraffin embedded (FFPE) tumours are the most readily available tissue samples and DNA derived from FFPE tissue has been validated for array comparative genomic hybridisation (aCGH) and single nucleotide polymorphism (SNP) array analysis. Furthermore, in the study of tumour precursor genetics, whole genome amplification (WGA) has been used to produce a sufficient amount of DNA for aCGH. However, it is unclear whether the same approach can be extended to high-resolution SNP analysis.
In this study, we examined the utility and limitations of genotyping platforms performed on WGA DNA from FFPE mesenchymal tumour samples for both copy number and SNP analyses. We analysed the results obtained using DNA derived from matched FFPE and frozen tissue samples on the Affymetrix 250K Nsp SNP array. Two widely used WGA methods, Qiagen (isothermal protocol) and Sigma (thermocycling protocol) were employed to determine how WGA methods affect the results.
We found that the use of WGA DNA derived from FFPE mesenchymal tumours for high-resolution SNP array application can produce a significant amount of false positive and false negative findings. While some of these misinterpretations appear to cluster in genomic regions with high or low GC contents, the majority appears to occur randomly. Only large scale chromosome loss of heterozygosity (LOH) (>10 Mb) can be reliably detected from WGA FFPE tumour DNA samples but not smaller LOH or copy number alterations.
Our findings here indicate a need for caution in SNP array data interpretation when using WGA FFPE tumour-derived DNA in determining genomic alterations less than 10 Mb.
在肿瘤遗传学研究中，福尔马林固定、石蜡包埋（FFPE）肿瘤是最容易获得的组织样本，并且已经验证了从 FFPE 组织中提取的 DNA 可用于阵列比较基因组杂交（aCGH）和单核苷酸多态性（SNP）阵列分析。此外，在肿瘤前体遗传学研究中，全基因组扩增（WGA）已被用于产生足够量的 DNA 进行 aCGH。然而，尚不清楚是否可以将相同的方法扩展到高分辨率 SNP 分析。
在这项研究中，我们研究了从 FFPE 间充质肿瘤样本的 WGA DNA 上进行基因分型平台的效用和局限性，用于进行拷贝数和 SNP 分析。我们使用来自匹配的 FFPE 和冷冻组织样本的 DNA，在 Affymetrix 250K Nsp SNP 阵列上分析了获得的结果。我们使用了两种广泛使用的 WGA 方法，Qiagen（等温协议）和 Sigma（热循环协议），以确定 WGA 方法如何影响结果。
我们发现，使用源自 FFPE 间充质肿瘤的 WGA DNA 进行高分辨率 SNP 阵列应用可以产生大量的假阳性和假阴性结果。虽然这些误判中的一些似乎聚集在 GC 含量高或低的基因组区域，但大多数似乎是随机发生的。只有大规模的杂合性丢失（LOH）（>10 Mb）可以从 WGA FFPE 肿瘤 DNA 样本中可靠地检测到，但较小的 LOH 或拷贝数改变则不能。
我们的研究结果表明，在使用源自 WGA FFPE 肿瘤的 DNA 确定小于 10 Mb 的基因组改变时，需要在 SNP 阵列数据解释中谨慎行事。
