Clinical Signifcance Of P53 Gene Mutation In Circulating Plasma DNA From Hepatocellular Carcinoma Patients

AIM: To evaluate the presence of tumor DNA in plasma from HCC(hepatocelluar carcinoma) and the possibility to be amplified by PCR. At the same time analyzed the clinical signifance of p53 gene mutation in plasma DNA . The experimental goal was to explore a new method for mutation screening and detection. In order to find a new alterative way for tumor diagnosis or monitoring . METHODS: 36 HCC (20 HBsAg+), 25 healthy blood donors ,21 HBV hepatitis/cirrhosis and 4 other begnign liver disease patients were enrolled in the study. DNA was extracted from plasma by using microcentrifugation column (Qiagen)and the-7-target DNA fragments(Exon5&6, 7 and 8)were amplified by PCR, The production was analyzed by DHPLC(denaturing high performance liquid chromatography), and the mutation status of p53 gene in circulating plasma DNA was finally confirmed by Sanger Di-deoxy termination method and direct DNA secquencing.RESULTS: Fragments of exons 5&6 were obtained from 10 of 36 HCC patients , while exon 7 and exon 8 were amplified from all of 36 HCC. But exons 5&6 obtained from none of 25 other liver disease patients, only 6 and 4 fragments of exon7 and exonS were obtained form this control group. But we did not get even one fragment from 25 health donors. 16( 12 of E7 ,4 of E5&6) and 2 mutation( 2 of E7 ) were detected by DHPLC from HCC and hepatitis/cirrhosis respectively. 2 HCC patients have 2 mutations, and 12(10 HCC) of 16 mutation patients was HBsAg+. CONCLUSION: Plasma DNA can be amplified more frequently in HCC,and small target fragment ( <200bp) is more efficiency, which may be caused by poor plasma DNA, DHPLC can be used to screen mutation in plasma DNA amplified fragements.p53 gene mutation can be found in hepatitis/cirrhosis but more frequently in HBsAg+ HCC(P <0.05).Detection mutation in plasma DNA suggests the clinical use in malignant diseases diagnosis and monitoring.