Takahashi Hiroshi, Sugai Tamotsu, Habano Wataru, Nakamura Shin-ichi
Division of Pathology, Iwate Medical University, Japan.
Pathol Int. 2002 Oct;52(10):628-35. doi: 10.1046/j.1440-1827.2002.01395.x.
The crypt isolation technique (CIT) allows for the isolation of pure tumor crypts from colon tumor tissue. In a previous study we reported on the genetic alterations found in colorectal tumor crypts using the CIT; however, a direct comparison of the genetic alterations found in colorectal carcinomas using either conventional methods (CM) or the CIT has not previously been performed. Here, we analyzed the impact of this method on the genetic analysis of colon tumor cells by comparing the observed frequency of genetic alterations in colon tumors isolated using CM or the CIT. We used a combination of the CIT and the fluorescent polymerase chain reaction assay to accurately assess the incidence of allelic imbalances (AI) at a number of chromosomal loci (17p, 5q, 18q, 1p, 8p, 22q), microsatellite instability (MSI), and mutations of cancer-related genes (p53 and APC genes) in 48 sporadic colorectal carcinomas. In addition, genetic alterations seen in multiploid tumors (defined as tumors with both diploid and aneuploid cell populations) identified by the CIT were also examined. The incidence of AI at the chromosomal loci tested was more frequently detected in samples isolated from tumors using the CIT than in those isolated from the same tumors using CM. In contrast, we observed no differences in the frequency of MSI or cancer-related gene mutation between the two groups. Although there was no difference in the frequency of genetic alterations between tumors with evidence of multiploidy, sorting of diploid and aneuploid populations allowed detection of distinct genetic changes. The crypt isolation method thus appears to be useful in that it allows purification of tumor cells and the accurate assessment of their genetic alterations. In addition, it may also be of benefit in clarifying the genetic profile of multiploid tumor cell populations.
隐窝分离技术（CIT）可从结肠肿瘤组织中分离出纯肿瘤隐窝。在之前的一项研究中，我们报告了使用CIT在结直肠肿瘤隐窝中发现的基因改变；然而，此前尚未对使用传统方法（CM）或CIT在结直肠癌中发现的基因改变进行直接比较。在此，我们通过比较使用CM或CIT分离的结肠肿瘤中观察到的基因改变频率，分析了该方法对结肠肿瘤细胞基因分析的影响。我们结合使用CIT和荧光聚合酶链反应分析，准确评估了48例散发性结直肠癌中多个染色体位点（17p、5q、18q、1p、8p、22q）的等位基因失衡（AI）发生率、微卫星不稳定性（MSI）以及癌症相关基因（p53和APC基因）的突变情况。此外，还检查了通过CIT鉴定的多倍体肿瘤（定义为具有二倍体和非整倍体细胞群的肿瘤）中出现的基因改变。在使用CIT从肿瘤中分离的样本中，测试的染色体位点处AI的发生率比使用CM从同一肿瘤中分离的样本中更频繁地被检测到。相比之下，我们观察到两组之间MSI或癌症相关基因突变频率没有差异。尽管有证据表明存在多倍体的肿瘤之间基因改变频率没有差异，但对二倍体和非整倍体群体进行分选可以检测到不同的基因变化。因此，隐窝分离方法似乎很有用，因为它可以纯化肿瘤细胞并准确评估其基因改变。此外，它在阐明多倍体肿瘤细胞群体的基因特征方面可能也有益处。
