Zhao Jianxin, Wu Rina, Au Alfred, Marquez Abbey, Yu Yibing, Shi Zuorong
Center for Biomedical Laboratory Science, San Francisco State University, California, USA.
Mod Pathol. 2002 Jun;15(6):657-65. doi: 10.1038/modpathol.3880582.
To compare the efficacy of chromogenic in situ hybridization (CISH(TM)) with fluorescence in situ (FISH) hybridization and immunohistochemistry (IHC) in determination of the HER2 status in human breast cancer.
HER2 gene amplification was determined on formalin-fixed paraffin-embedded (FFPE) sections of 62 invasive breast cancers by FISH and followed by CISH using a digoxigenin (DIG)-labeled HER2 DNA probe generated by Subtraction Probe Technology (SPT(TM)), and a biotin-labeled chromosome 17 centromeric (chr.17cen) probe. The sections were heat treated and enzyme digested. After in situ hybridization, the HER2 probe was detected with fluorescein (FITC)-anti-DIG for FISH, followed by peroxidase-anti-FITC and diaminobenzidine (DAB) for CISH. The chr.17cen probe was detected with peroxidase-streptavidin and DAB. For CISH application, HER2 gene copies or chromosome 17 centromeres and morphology of cells were easily visualized simultaneously with a 40x objective under bright-field microscope in hematoxylin-counterstained sections. IHC study of HER2 overexpression was performed on adjacent sections using a panel of three HER2 antibodies (TAB 250, CB11, A0485), and staining was scored according to the criteria specified in the HercepTest.
HER2 gene amplification detected by CISH was visualized typically as large DAB-stained clusters or by many dots in the nucleus. FISH and CISH identified HER2 gene amplification in 19% of the tumors. Chromosome 17 polysomy was detected in 31% of the tumors. HER2 overexpression was demonstrated in 19% (TAB 250), 23% (CB11), and 36% (A0485) of the tumors. Complete concordance between the results of CISH with FISH, TAB 250, CB11, and A0485 was seen in 100%, 97%, 94%, and 84% of the cases, respectively.
By permitting observation of morphology using a bright-field microscope, CISH is an accurate, practical, and economical approach to screen HER2 status in breast cancers. It is a useful methodology for confirming ambiguous IHC results.
比较显色原位杂交（CISH™）与荧光原位杂交（FISH）及免疫组织化学（IHC）在检测人乳腺癌HER2状态方面的疗效。
采用FISH对62例浸润性乳腺癌的福尔马林固定石蜡包埋（FFPE）切片进行HER2基因扩增检测，随后使用通过减法探针技术（SPT™）生成的地高辛（DIG）标记的HER2 DNA探针和生物素标记的17号染色体着丝粒（chr.17cen）探针进行CISH检测。切片经热处理和酶消化。原位杂交后，FISH用异硫氰酸荧光素（FITC）-抗DIG检测HER2探针，随后CISH用过氧化物酶-抗FITC和二氨基联苯胺（DAB）检测。chr.17cen探针用过氧化物酶-链霉亲和素和DAB检测。对于CISH应用，在苏木精复染切片中，在明场显微镜下用40倍物镜可轻松同时观察到HER2基因拷贝或17号染色体着丝粒以及细胞形态。使用一组三种HER2抗体（TAB 250、CB11、A0485）在相邻切片上进行HER2过表达的IHC研究，并根据HercepTest规定的标准对染色进行评分。
CISH检测到的HER2基因扩增通常表现为大的DAB染色簇或细胞核中的许多小点。FISH和CISH在19%的肿瘤中检测到HER2基因扩增。在31%的肿瘤中检测到17号染色体多体性。在19%（TAB 250）、23%（CB11）和36%（A0485）的肿瘤中证实有HER2过表达。CISH与FISH、TAB 250、CB11和A0485结果的完全一致性分别在100%、97%、94%和84%的病例中可见。
通过允许使用明场显微镜观察形态，CISH是一种准确、实用且经济的筛查乳腺癌HER2状态的方法。它是确认模糊IHC结果的有用方法。
