The Identification Of Gene Mutation Profiling In Primary Liver Cancer And The Analysis Of Expression Pattern Of Differentiation-related Genes

Primary liver cancers include hepatocellular carcinoma(HCC), intrahepatic cholangiocarcinoma (ICC), combined hepatocholangiocarcinomas (cHCC-CC) and some other rare types. AFP is the unique protein in HCC. HCC patients are commonly with high expression level of AFP, but the concentration of AFP in serum of some patients is no more than20ng/ml. And those HCCs with low AFP are defined as AFP-negative HCCs. Primary intrahepatic cholangiocarcinoma (ICC) is another type of liver tumor second to HCC. It is different from extrahepatic bile duct carcinoma and hilarcholangicarcinoma, accounting for5%-20%of all primary malignant liver tumors.Section I, to identify the genetic pathogenesis of AFP-negative HCC and ICC, we adopted exome capture-based SOLiD high-throughput sequencing platform to analyze somatic mutations of coding sequences in the19-paired AFP-negative HCC patients and12-paired ICC patients. Those coding DNA were enriched by exome capture technique from genomic DNA of patients specimens, then their sequences were resolved by SOLiD sequnceing platform. High-confidence, medium confidence and low confidence candidate somatic mutations were identified from high-throughput sequence reads under different filtering parameters, respectively. To validate those candidate somatic mutations and determine mutated genes, some candidate mutations were subject to PCR-Sanger convention sequencing. As a result, the accurate rate of high-confidence mutation in those AFP-negative HCC patients is55.6%, and the accurate rate of low-confidence mutation is30.8%; The results demonstrate the identification of somatic mutations based on high-throughput sequencing is feasible.Section II, to investigate differentiation characteristics and mutaions of a known HCC-causing gene TP53in these AFP-positive and AFP-negative HCCs, we analyzed the coding and splice sequences of TP53gene in62-paired HCC patients. The results show that TP53gene mutations occurred in30%HCC specimens examined. In addition, we examined expression characteristics of early markers gene of liver cancer AFP、DLK1、EPCAM、ALB、 CK18、CK19and transcription factor gene of embryonic stem cell NANOG、OCT4、SOX2in HCC samples. The results show that DLK、EPCAM have significantly difference in AFP-negative HCC and AFP-positive HCC, suggesting that the differentiation degree of AFP-positive HCC is low and can express gene of DLK、EPCAM. Significantly, we also find that transcription factor of embryonic stem cell OCT4and NANOG have highly correlation in these samples.