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

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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.