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大肠癌原发灶和肝转移灶p53变异及蛋白表达
了解p53基因及其蛋白在大肠癌发生、转移过程中的动态变化。方法 p53基因外显子5～9以DGGE及自动DNA序列分析来检测，p53蛋白表达以免疫组化方法检测。结果 34例中21例呈p53变异，占61.8%(包括2例PCR失败但免疫染色阳性者)，其中5例仅在肝转移灶发现p53变异，其余均为原发灶、转移灶一致性的变异。另有2例原发灶即有p53变异者，在转移灶出现了新增加的变异。在37处变异中，27处为错义突变，占73.0%；6处为无义突变，占16.2%；4处微小插入。在原发、转移灶同时有单个碱基改变的14例中，13例呈现变异的p53碱基峰和正常峰之比，在肝转移灶明显高于大肠原发灶(P＜0.001)。p53免疫组化染色结果和DGGE、DNA序列分析结果高度一致。但4例(6处)呈无义突变的病例，免疫组化均为阴性。另有3例在DGGE和序列分析中无法证明任何p53变异的大肠癌原发灶，免疫组化显示p53蛋白的过度表达。结论 在大肠癌肝转移过程中，p53变异主要开始于肠癌原发灶，并被保持于转移至肝脏的癌细胞内，在转移灶其含量或含变异p53的癌细胞量明显增加。小部分p53变异也可以始发于转移灶。p53变异与p53蛋白过度表达呈正相关。但单独应用p53基因检测或免疫组化技术均可导致假阴性的出现。
p53 mutations and protein overexpression in primary colorectal cancer and its liver metastasis YANG Yang, XIA Qiong, LIAN Fangjie, et al. Department of Oncology, Nanfang Hospital, Guangzhou 510515
【Abstract】 Objective To compare p53 status between primary and hepatic metastatic colorectal cancer in 34 patients.Methods p53 gene status (exon 5-9) was examined by PCR, denaturing gradient gel electrophoresis (DGGE) and automated sequencing. p53 protein was detected by immunohistochemistry using monoclonal antibody DO-7.Results p53 mutations were found in exons 5 through 9 in 21 of 34 patients (61.8%). Among them, 5 patients had mutation in liver metastasis but not in their primary tumor while in the other 4 patients the same mutations were found in both primary and metastatic colorectal cancers. In no patients was p53 mutation exclusively found in the primary colorectal tumor. Moreover, additional mutation was detected in the metastatic lesions in two cases. Of the 37 mutations within the exons examined, 73% was missense mutation and 16% was nonsense mutation. There were 4 microinsertions. p53 protein was overexpressed in both primary and metastatic colorectal cancers with p53 gene mutations. The presence of p53 mutations significantly correlated with p53 protein accumulation (r=0.96, P＜0.001). However, in 4 patients with p53 nonsense mutation, immunohistochemical staining was negative. In three patients who showed no p53 mutation of the primary tumor, p53 protein was consistently over-expressed.Conclusion In colorectal cancer, p53 gene mutation usually appears first in the primary tumor and maintains as such but more prominent when metastasized to the liver. However, p53 gene mutation may occur only after being metastasized. Although p53 gene mutation and p53 protein overexpression correlate with each other, either parameter examined alone may lead to false positive results.
【Subject words】 Colorectal neoplasms Liver neoplasms/secondary p53 gene p53 protein
关于p53与大肠癌及其肝转移的研究已有很多［1，2］，但尚未见在同一患者个体水平上观察比较其原发灶和转移灶p53状态的报道。我们选择了34例大肠癌术后并发肝转移者，应用DGGE(denaturing gradient get electro-phoresis)和自动DNA序列分析技术，对每一个体的原发病灶和肝转移灶的p53状态进行检测并比较，以了解在癌瘤发生、发展、转移过程中p53基因的改变及其意义。
