A microdissection approach to detect molecular markers during progression of prostate cancer.
A microdissection approach to detect molecular markers during progression of prostate cancer.
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DOI：
10.1038/bjc.1995.439
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发表时间：
1995-10
影响因子：
8.8
通讯作者：
Maitland, N J
中科院分区：
文献类型：
--
作者：
Berthon, P;Dimitrov, T;Stower, M;Cussenot, O;Maitland, N J
为了研究癌变的潜在机制，我们开发了一种技术来确定前列腺癌组织中遗传变化的频率。我们已经证明，在1：4和1：9之间的比例突变正常等位基因，从突变TP 53等位基因的信号是不明显的聚合酶链反应（PCR）扩增和进一步的直接测序或单链构象多态性（SSCP）分析。为了绕过这个问题，这是固有的异质性的前列腺组织和肿瘤，我们选择了区域的分级前列腺肿瘤（格里森评分）从冷冻切片制备和显微解剖这些细胞（20-100个细胞）。经阴离子交换树脂去除蛋白质，PCR扩增TP 53基因外显子5/6，SSCP分析后，在可疑肿瘤细胞中观察到异常SSCP条带移动，与作为内部对照的显微切割的基质细胞相比，而（1）携带肿瘤的组织DNA的粗制品没有显示任何异常，和（2）用一组抗TP 53蛋白的单克隆抗体进行的免疫染色仍为阴性。不同条带的核苷酸序列分析证实了一份标本的异常条带中TP 53基因外显子6位置13，336处存在突变，而在正常SSCP条带中未检测到突变。通过靶向识别的肿瘤细胞，我们可以发现使用全组织DNA提取的标准技术检测不到的DNA突变，特别是在异质性肿瘤如前列腺癌中。
To investigate the underlying mechanisms of carcinogenesis, we have developed a technique to determine the frequency of genetic changes in prostatic carcinoma tissue. We have demonstrated that at a ratio of between 1:4 and 1:9 mutant-normal alleles, the signal from a mutant TP53 allele is not apparent after polymerase chain reaction (PCR) amplification and further direct sequencing or single-strand conformation polymorphism (SSCP) analysis. To bypass this problem, which is inherent in the heterogeneity of the prostate tissue and of the tumour, we selected areas of graded prostate tumours (Gleason score) from cryosectioned preparations and microdissected these cells (20-100 cells). After anionic resin removal of proteins, PCR amplification of TP53 gene exons 5/6 and SSCP analysis, an abnormal SSCP band shift was observed in suspected tumour cells, compared with microdissected stromal cells used as an internal control, while (1) a crude preparation of tissue DNA carrying the tumour did not show any abnormality and (2) immunostaining by a set of monoclonal antibodies against TP53 protein remained negative. Nucleotide sequence analysis of the different bands confirmed the presence of a mutation in the TP53 gene exon 6 position 13,336 in an abnormal band for one specimen, while no mutation was detected in the normal SSCP band. By targeting recognised tumour cells we can find DNA mutations which are undetectable using the standard technique of whole-tissue DNA extraction, particularly in a heterogeneous tumour such as carcinoma of the prostate.