Predictive Value Of The G1-G6 Transcriptomic Molecular Classification Of Hepatocellular Carcinoma For Its Biological Behavior And Clinicopathological Features

ObjectiveTo explore the predictive value of the G1-G6 transcriptomic molecular classification of hepatocellular carcinoma(HCC)for its biological behavior and clinicopathological characteristics in Chinese patients,focusing on prognosis risk factors.Then find out the groups with the high mortality rate from HCC patients and make the individualized treatment strategies to decrease the risk of death and recurrence.Methods1.A total of 107 HCC patients who received partial hepatectomy without any treatment before at Mengchao Hepatobiliary Hospital of Fujian Medical University from January2014 to December 2017 were collected for a retrospective cohort study.They met the inclusion criteria and had detailed clinicopathological data and follow-up information.The expression of 16 predictor genes related to the G1-G6 molecular typing in patients was detected,and the results were obtained by taking them into the mathematical model constructed by Boyault et al.2.Clinicopathological data and follow-up information of HCC patients were collected.Chi-square test was used to analyze the correlation between the G1-G6 molecular typing and tumor biological behavior,patients’ clinical characteristics,pathological typing,and serum marker level.Kaplan-Meier and Cox proportional hazards survival regression analysis was used to evaluate the G1-G6 molecular classifications’ prognostic significance.3.According to the molecular classification,58 paraffin-embedded tissue samples from HCC patients were randomly selected using a stratified sampling procedure to make tissue microarrays.4.Quantitative real-time polymerase chain reaction(qPCR),western blot(WB),immunohistochemistry(IHC),and other methods were used to investigate the expression of 16 predicted genes,programmed death-ligand 1(PD-L1),Wnt/β-catenin signaling pathway markers,epithelial-mesenchymal transition(EMT)markers,and liver cancer cell stemness markers in patients’ tumor and adjacent liver tissues to verify the applicability of the G1-G6 molecular classification in Chinese and evaluate its clinical significance.5.RNA sequencing(RNA-seq),bioinformatics analysis,and the cancer genome atlas(TCGA)database analysis were employed to investigate the target genes and molecular signaling pathways of liver cancer’s postoperative recurrence.Results1.HCC from China(n=107)were distributed(%)into G1(17.76),G2(1.87),G3(18.69),G4(9.35),G5(23.36),and G6(28.97)groups.2.It was demonstrated that the G1-G6 molecular classification was significantly correlated with tumor number,serum alpha fetoprotein(AFP)level,hepatitis B virus(HBV)DNA copy number,liver cirrhosis,and microvascular invasion(MVI).Moreover,G1 was associated with high serum AFP level(P=0.001);G3 was associated with a nonsingle trabecular histological structure(P=0.021)and high copy number of HBV DNA(P=0.014);G4 was associated with advanced age(P=0.012)and low copy number of HBV DNA(P=0.012);G5 was associated with single tumor(P=0.013)and low incidence of MVI(P=0.007);G6 was associated with strong positive Hep-Par1(P=0.007).Further statistical analysis found that the proliferation group(G1-G3 group)was associated with high serum AFP level(P=0.002),high HBV DNA copy number(P=0.047),a non-single trabecular histological structure(P=0.010),macrovascular invasion(P=0.012),and HepPar1 negative or weakly positive(P=0.012).3.Kaplan-Meier survival analysis indicated that HCC patients in G1-G3 group had shorter overall survival time than that in G4-G6 group within three years(P=0.0102),and G3 subgroup has the worst prognosis(P=0.0125).Cox regression analysis showed that TNM staging(P=0.0002),BCLC staging(P=0.001),and the G1-G6 molecular classification(P=0.012)were significant factors affecting the overall prognosis of HCC patients.Multivariate analysis showed that the G1-G6 molecular classification(P=0.035)was an independent prognostic risk factor for poor overall survival(OS)of HCC patients.4.It was found that the expression levels of AFP(P=0.0200),cadherin 2(CDH2)(P=0.0022),Jupiter microtubule associated homolog 1(HN1/JPT1)(P<0.0001),NRAS(P=0.0096),p21 activated kinase 2(PAK2)(P= 0.0024),RAB1A(P=0.0020),and SUMO1 activating enzyme subunit 1(SAE1)(P<0.0001)in G1-G3 group were significantly higher than in G4-G6 group by qPCR.Conversely,the expression levels of laminin subunit alpha(LAMA3)(P=0.0059)and regenerating family member 3 alpha(PAP/REG3A)(P=0.0495)in G4-G6 group were markedly higher than in G1-G3 group.5.PD-L1 SP142,28-8,and 73-10 were detected via IHC.The results showed that G1-G3 group had higher PD-L1 protein expression than G4-G6 group.6.Using qPCR,WB,and IHC methods discovered that G5 and G6 subgroups were related to the activation of the Wnt/β-catenin pathway and EMT.Furthermore,we observed a high expression of stemness markers(Ep CAM and SOX9)in G1-G3 group,especially in G1 subgroup.7.By analyzing the transcriptome sequencing data of 12 HCC patients,we identified the differentially expressed genes between the proliferation group(G1-G3)and nonproliferation group(G4-G6)based on the G1-G6 signature,including regenerating family member 1 beta(REG1B),regenerating family member 3 gamma(REG3G)and inositol1,4,5-trisphosphate receptor type 1(ITPR1).Additionally,KEGG pathway enrichment analysis revealed that calcium signaling pathway,c GMP-PKG signaling pathway,renin secretion,neuroactive ligand-receptor interaction,and cell metabolism-related signaling pathways were significantly enriched in G1-G3 group.Conclusion1.The G1-G6 transcriptomic molecular classification is applicable in the Fujian HCC cohort regardless of patients’ ethnic origin and detecting m RNA levels by qPCR using SYBR green I instead of the Taq Man probe in the reaction.2.G1-G3 group can be used as an independent risk factor for evaluating HCC patients’ prognosis and is significantly correlated with poor prognosis after hepatectomy,and G3 subgroup has the worst prognosis.3.It was been successfully verified that G5 and G6 subgroups were associated with the activation of the Wnt/β-catenin pathway.It is distinct from previous studies that we discovered G1-G3 group was associated with PD-L1 expression and activation of the calcium signaling pathway and G1 subgroup is mainly related to liver cancer stemness.4.Based on RNA-seq and TCGA database,it was found that ITPR1 with high expression in HCC is significantly correlated with poor prognosis and is a potential marker of HCC.