Section of Cancer Genomics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Anal Cell Pathol (Amst). 2010;33(2):105-12. doi: 10.3233/ACP-CLO-2010-0532.
The simultaneous detection of protein expression and gene copy number changes in patient samples, like paraffin-embedded tissue sections, is challenging since the procedures of immunohistochemistry (IHC) and Fluorescence in situ Hybridization (FISH) negatively influence each other which often results in suboptimal staining.Therefore, we developed a novel automated algorithm based on relocation which allows subsequent detection of protein content and gene copy number changes within the same cell.
Paraffin-embedded tissue sections of colorectal cancers were stained for CD133 expression. IHC images were acquired and image coordinates recorded. Slides were subsequently hybridized with fluorescently labeled DNA probes. FISH images were taken at the previously recorded positions allowing for direct comparison of protein expression and gene copy number signals within the same cells/tissue areas. Relocation, acquisition of the IHC and FISH images, and enumeration of FISH signals in the immunophenotyped tumour areas were done in an automated fashion.
Automated FISH analysis was performed on 13 different colon cancer samples that had been stained for CD133; each sample was scored for MYC, ZNF217 and Chromosome 6 in CD133 positive and negative glands. From the 13 cases four (31%) showed amplification for the MYC oncogene and seven of 13 (54%) cases were amplified for ZNF217. There was no significant difference between CD133 positive tumour and CD133 negative tumour cells.
The technique and algorithm presented here enables an easy and reproducible combination of IHC and FISH based on a novel automated algorithm using relocation and automated spot counting.
在石蜡包埋组织切片等患者样本中同时检测蛋白质表达和基因拷贝数变化具有挑战性，因为免疫组织化学（IHC）和荧光原位杂交（FISH）的程序相互影响，通常导致染色效果不佳。因此，我们开发了一种基于重定位的新型自动化算法，允许在同一细胞内后续检测蛋白质含量和基因拷贝数变化。
对结直肠癌的石蜡包埋组织切片进行 CD133 表达染色。获取 IHC 图像并记录图像坐标。随后用荧光标记的 DNA 探针对载玻片进行杂交。在先前记录的位置拍摄 FISH 图像，允许在同一细胞/组织区域内直接比较蛋白质表达和基因拷贝数信号。以自动化方式进行重定位、获取 IHC 和 FISH 图像以及对免疫表型肿瘤区域中的 FISH 信号进行计数。
对 13 个不同的结肠癌样本进行了自动 FISH 分析，这些样本均已对 CD133 进行了染色；对 CD133 阳性和阴性腺体中的 MYC、ZNF217 和 6 号染色体进行了评分。在 13 例中，有 4 例（31%）显示 MYC 癌基因扩增，7 例（54%）显示 ZNF217 扩增。CD133 阳性肿瘤细胞和 CD133 阴性肿瘤细胞之间没有显著差异。
本文介绍的技术和算法通过使用重定位和自动斑点计数的新型自动化算法，实现了 IHC 和 FISH 的轻松、可重复组合。
