The molecular and clinicopathological study related to detection of K-ras point mutation in the blood of pancreatic cancer cases

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Purpose : We have attempted to detect K-ras mutation from blood specimens in order to assist in the diagnosis of pancreatic cancer.Materials : The blood (perioperative peripheral blood and intraoperative portal blood) and tissues of the primary lesion in the pancreas were taken from 16 patients with pancreatic cancer (n=14) and benign diseases (n=2 ; chronic pancreatitis and insulinoma).Methods and Results :1.Detection of K-ras mutation in the primary lesionAfter microdissection we extracted DNA whose K-ras gene was amplified by PCR system. Its sequence was identified by digoxigenin dot blotting. The incidence of mutation at codon 12 was 85%.2.Detection of K-ras mutation from blood specimensBy the magnetic cell sorting system (MCSS) using anticytokeratine antibody and magnetic microspheres, we concentrated epithelial cells from the blood. Following amplification of the K-ras gene we enriched the mutated gene by the PCR-RFLP method. The sensitivity of PCR-RFLP without MCSS was only 10^< … More -4>, whereas that of combination of PCR-RFLP and MCSS reached 10^<-6> -10^<-7> With this combined method we could detect K-ras mutation in all samples of the portal blood and postoperative peripheral blood when the patients had K-ras mutation in the primary tumor. Furthermore, the mutation was detected in 25% even in the preope-rative peripheral blood. These cases rapidly deteriorated after surgery and died 3-6 months postoperatively.Discussion and ConclusionThe current study showed that tumor cells existed in the peripheral blood of pancreatic cancer patients at the rate of one tumor cell per 105 to 107 normal nucleated cells. We conjectured that the number of circulating cancer cells in the preoperative blood might be several thousand per body, and that the operative injury seemed to increase their number ten times. It was suggested that prognosis would be extremely poor if patients had tens of thousand of circulating cancer cells in the peripheral blood before surgey. We think that even the combination of PCR-RFLP and MCSS is not appropriate for early detection of pancreatic cancer. However, this system appears to be useful in excluding contraindicated operative cases from surgery. Less