Analysis Of Genomic DNA Mutation And Diagnostic Value Of Circulating Tumor DNA In Hepatocellular Carcinoma

Objectives:Circulating tumor DNA(ctDNA)contains tumor-specific mutations and could potentially serve as "liquid biopsy".This study aims to screen the differentially expressed ctDNA in hepatocellular carcinoma(HCC)and evaluate the diagnostic values of these ctDNA.Materials and Methods:33 Liver tissue,37 blood,37 and swab specimens were collected from patients with HCC and control individuals(N=6).Their genomic DNA and ctDNA were subjected to next generation sequencing(NGS).The significantly mutated genes and pathway enrichment in the HCC versus control groups were analyzed,and the correlation between clinical phenotypes and mutated genes was analyzed.Finally,a group of candicate genes in ctDNA were selected and their diagnostic performance was evaluated.Results:Eleven genes carried significant mutation were identified by bioinformatic analysis in the study.Six genes(TP53,CTNNB1,AXIN1,JAK1,EPS15,and CACNA2D4)that were recognized as the most significantly mutated genes in HCC,and five genes(ARIDIA,FLCN,SETD2,PTEN,and BUB IB)that were well-known to be frequently mutated in other cancer types.We compared the selected mutaions of recurrent and non-recurrent patients,and found the mutation frequency was significantly higher in the patients with recurrent HCC.The result of pathway enrichment analysis of mutation genes showed that three signal pathways occurred significant mutations in HCC,including the cell cycle,PI3K/Akt,and Wnt signaling pathways.The patients with the mutations in the pathways had worse prognosis and higher risk of HCC recurrent than the patients without the mutations.Moreover,all HCC patients were catogarized into three subclasses(S1,S2 and S3)based on gene expression array data.The results showed the patients of S3 subclass had the longest survival while S2 subclass had the highest alpha-fetal protein(AFP)levels.Some new mutaions were indentified in three subclasses using genetic profiles.A majority of selected mutations in the HCC tissue DNA,ranging from 52%-84%,was detected in the matched plasma ctDNA.We selected 13 genes in ctDNA to diagnose HCC,including TP53,AXIN1,JAK1,CTNNB1,BRAF,EGFR,CDKN2A,PTEN,FGFR2,KDR,LONP1,MAP2K2 and SMAD2.For the candidate mutations,receiver operating characteristic(ROC)analysis revealed an area under the ROC curve(AUC)of 0.92,sensitivity of 65%,and specificity of 100%for the diagnosis of HCC.Detection of the selected mutations in ctDNA in combination with AFP levels exhibited better diagnosis performance,with AUC of 0.96,sensitivity of 73%,and specificity of 100%for Low-AFP patients,whereas the AUC was 0.86,with sensitivity of 53%and specificity of 100%for High-AFP patients.Conclusion:This study has identified the feature of genetic mutations and its relationship with clinical phenotypes in HCC.A novel panel of candidate genes were defined to detect the driver mutations in ctDNA.Furthermore,combinated with AFP levels,the mutations of candidate genes in ctDNA showed better diagnostic performance.