Savola S, Nardi F, Scotlandi K, Picci P, Knuutila S
Department of Pathology, Haartman Institute, University of Helsinki, Finland.
Cytogenet Genome Res. 2007;119(1-2):21-6. doi: 10.1159/000109614. Epub 2007 Dec 14.
Deletion of the CDKN2A locus at 9p21.3 has been reported to be a poor prognostic sign in the Ewing sarcoma family of tumours. In clinical applications CDKN2A deletion is primarily detected using fluorescent in situ hybridisation (FISH) with a commercial probe, size approximately 190 kb. Due to limitations in resolution, FISH analysis may fail to detect microdeletions smaller than 190 kb. In the present study, we performed 44K array comparative genomic hybridisation (CGH) on eleven Ewing sarcoma cell lines and 26 tissue samples in order to define the sizes of 9p21.3 deletions. Microarray CGH analysis revealed 9p21.3 deletions encompassing the CDKN2A locus in eight cell lines (73%) and in six tumours (23%). In four cases (two cell lines and two tissue samples) the deletion was less than 190 kb in size. In one cell line sample, we detected a microdeletion of approximately 58 kb in 9p21.3 harbouring the CDKN2A locus. We confirmed this result using 244K microarray CGH and TaqMan quantitative RT-PCR analysis and further performed FISH analysis on this cell line sample. Here, we show that CDKN2A FISH analysis can give false negative results in cases with small microdeletions. Our results suggest that new and more accurate FISH methods should be developed for detection of deletions in the CDKN2A locus.
据报道，9p21.3处CDKN2A基因座的缺失在尤因肉瘤家族性肿瘤中是预后不良的标志。在临床应用中，CDKN2A缺失主要通过使用商业探针的荧光原位杂交（FISH）检测，探针大小约为190 kb。由于分辨率的限制，FISH分析可能无法检测到小于190 kb的微缺失。在本研究中，我们对11个尤因肉瘤细胞系和26个组织样本进行了44K阵列比较基因组杂交（CGH），以确定9p21.3缺失的大小。微阵列CGH分析显示，8个细胞系（73%）和6个肿瘤（23%）中存在包含CDKN2A基因座的9p21.3缺失。在4例（2个细胞系和2个组织样本）中，缺失大小小于190 kb。在1个细胞系样本中，我们在9p21.3中检测到一个约58 kb的微缺失，该区域包含CDKN2A基因座。我们使用244K微阵列CGH和TaqMan定量RT-PCR分析证实了这一结果，并对该细胞系样本进一步进行了FISH分析。在这里，我们表明，在存在小微缺失的情况下，CDKN2A FISH分析可能会给出假阴性结果。我们的结果表明，应该开发新的、更准确的FISH方法来检测CDKN2A基因座的缺失。
