Detection of tumor cells in peripheral blood by PCR-SSCP using the oncogene as DNA marker

早期发现血源性转移对改善患者预后具有重要意义。在这项研究中，我们评价了改良的PCR-SSCP方法检测胃癌和结直肠癌患者循环血中癌细胞的方法。以P53抑癌基因和K-ras癌基因作为DNA标记物，发现这些癌组织中存在高频基因突变。由于循环血中肿瘤细胞数量很少，我们从一例原发肿瘤的局部静脉采血，利用抗CD45单抗的磁性细胞分离系统对肿瘤细胞进行富集化。在该方法中，肿瘤细胞与正常细胞的比例增加了20倍。此外，我们还尝试了首次聚合酶链式反应后的套式聚合酶链式反应，大大提高了检测水平，可以检测到相当于一个细胞的DNA。用这种改进的SSCP方法检测了3例胃癌和2例结直肠癌患者的原发肿瘤标本和血液标本。其中1例在原发肿瘤标本和血液标本中显示相同的突变条带。为了通过SSCP证实该突变的特异性，我们用Southern杂交、直接测序和菌落杂交技术进行了检测。SSCP检测到的突变条带为K-ras密码子12由Gly(GGT)突变为Asp(GAT)的单点突变。该方法灵敏度高，可在1000个正常细胞中检测到约1.2个肿瘤细胞。因此，我们认为这是一种非常有用的检测循环血液中癌细胞的方法。

The early detection of haematogeneous metastasis is very important for improvement of prognosis in patients. In this study, we evaluated the improved PCR-SSCP method for detection of cancer cells in circulation blood in patients with gastric and colorectal cancer. As a DNA marker, we used the p53 tumor suppressor gene and K-ras oncogene which shows the high frequency gene mutation in these cancer. As the number of tumor cells in circulation blood is very small, we took blood samples from the local vein of a primary tumor, and enriched the tumor cells by a magnetic cell separation system which used the anti-CD45 monoclonal antibody. In this method, the ratio of tumor cells in normal cells was enriched twenty times. Futhermore, we tried the nested-PCR following the first PCR.The level of detection greatly rose and DNA equivalent to one cell could be detected.We examined the primary tumor sample and blood sample in patients with gastric cancer (3cases) and colorectal cancer (2cases) by this improved SSCP method. One of these 5 cases, showed the same mutation band in the primary tumor sample and blood samples. To confirm the specificity of this mutation by SSCP,we examined the southern blot analysis, direct sequence and colony hybridization techniques. The mutation band by SSCP was certainly a one point mutation of K-ras codon 12 from Gly (GGT) to Asp (GAT). This method is very sensitive and it is possssible to detect about 1.2 tumor cell in 1000 normal cells. Therefore, we think this is a very useful method for detection of cancer cells in circulating blood.