Lee W, Han K, Harris C P, Meisner L F
University of Wisconsin, State Laboratory of Hygiene, Madison 53706.
Cancer Genet Cytogenet. 1993 Jul 15;68(2):99-103. doi: 10.1016/0165-4608(93)90004-6.
Conventional cytogenetic studies of solid tumors are limited by the difficulty of culturing tumor cells, while in situ hybridization using paraffin sections of interphase cells results in too many truncated cells. To solve these problems, fluorescent in situ hybridization (FISH) technique was used on free nuclei isolated from formalin-fixed paraffin-embedded embryonal rhabdomyosarcoma (RMS) tissue using our modification of Hedley's method for isolation of nuclei. Biotinylated DNA probes for the centromeric regions of chromosomes 6, 8, 11, 12, 17, and 18, painting probes for chromosomes 8 and 11, and a cosmid probe for the HER-2/neu oncogene, were used. The centromeric probes worked well, demonstrating two copies of chromosomes 6, 17, and 18, but three copies of chromosome 11 in 52.9% of nuclei. Four copies of chromosome 8 were observed in 57.1% of nuclei and five or more in 17.1%. Chromosome 12 demonstrated 21.8% trisomy and 62.2% tetrasomy. Painting probes for chromosome 11 also worked well and matched the results of the centromeric probes, with no suggestion of structural aberration. However, the results of the painting probe for chromosome 8 yielded fluorescent areas of different sizes, suggesting that some of the extra chromosomes 8 could be deleted. The cosmid probe for the HER-2/neu oncogene also worked well, and revealed two signals in each nucleus without evidence of amplification. This study illustrates the successful use of a new technique for studying chromosomal aberration in paraffin-embedded solid tumors. The importance of this technique is that it has not been previously possible to use painting probes or cosmid probes on paraffin tissue sections. Use of this procedure will broaden the type of retrospective studies that can be performed to include detection of deletions or translocations.
实体瘤的传统细胞遗传学研究受到肿瘤细胞培养困难的限制，而使用间期细胞石蜡切片进行原位杂交会产生过多截断细胞。为了解决这些问题，我们采用了对赫德利细胞核分离方法的改进，对从福尔马林固定石蜡包埋的胚胎性横纹肌肉瘤（RMS）组织中分离出的游离细胞核进行荧光原位杂交（FISH）技术。使用了针对6号、8号、11号、12号、17号和18号染色体着丝粒区域的生物素化DNA探针、针对8号和11号染色体的描绘探针以及针对HER-2/neu癌基因的黏粒探针。着丝粒探针效果良好，显示在52.9%的细胞核中6号、17号和18号染色体有两个拷贝，但11号染色体有三个拷贝。在57.1%的细胞核中观察到8号染色体有四个拷贝，在17.1%的细胞核中有五个或更多拷贝。12号染色体显示21.8%三体和62.2%四体。11号染色体的描绘探针也效果良好，与着丝粒探针的结果相符，未提示结构畸变。然而，8号染色体描绘探针的结果产生了大小不同的荧光区域，表明一些额外的8号染色体可能发生了缺失。HER-2/neu癌基因的黏粒探针也效果良好，每个细胞核显示两个信号，无扩增证据。本研究说明了一种用于研究石蜡包埋实体瘤染色体畸变的新技术的成功应用。该技术的重要性在于此前无法在石蜡组织切片上使用描绘探针或黏粒探针。使用该程序将拓宽可进行的回顾性研究类型，包括检测缺失或易位。
