Intratumor Heterogeneity Of Hepatocellular Carcinoma And Research On The Mechanism Of Its Association With Prognosis

Background&Aims:Hepatocellular carcinoma(HCC)is heterogeneous,which decreases the efficacy of clinical treatments.Understanding tumor heterogeneity of HCC is critical when developing novel treatment strategies.However,a comprehensive investigation of tumour heterogeneity in HCC is limited.Besides,few studies have explored the association between genetic intratumor heterogeneity and patient prognosis in HCC.In this study,we will analyze intratumor heterogeneity of HCC comprehensively from the aspect of pathology,genome,transcriptome and immune microenvironment.Furthuremore,we plan to explore the association between genetic intratumor heterogeneity and prognosis,and to study the potential mechanism behind this association.Methods:We harvested 25 tumor samples from nine HCC patients by multi-region sampling strategy.Then we performed hematoxylin-eosin(HE)staining,whole-exome sequencing,RNA sequencing,immunohistochemistry and flow cytometry on all samples,evaluating intratumor heterogeneity from the aspect of pathology,genome,transcriptome and immune microenvironment.Furthermore,by analyzing clinical information of these patients,we explored the association between genetic intratumor heterogeneity and prognosis,and then used AJCC Ⅰ stage HCC cohorts from TCGA as validation.Besides,we also compared the difference of RNA expression,immune infiltrates and neoantigens between genetic intratumor heterogeneity group of various levels,in order to reveal the mechanism behind the association between intratumor heterogeneity and prognosis.Results:There are various levels of intratumour heterogeneity with regard to the histology,genomes,transcriptomes,and tumor infiltrating immune cells.A potential association between genetic mutation,histology and immune cells in different regions of the tumor was also seen.In 7 BCLC A stage HCC patients,the group with high intratumor heterogeneity index showed the tendency of having higher recurrence free survival rate.Furthermore,in AJCC Ⅰ stage HCC cohorts from TCGA,the group with high intratumor heterogeneity were associated with higher disease specific survival rate and progression free survival rate(p = 0.0176,0.0360).Data from tumor multiregion sampling showed that tumors with high intratumor heterogeneity had more infiltration of CD8 T cells(p=0.0204),CD4 T cells(p = 0.0114),B cells(p = 0.0011)and higher cytolytic activity(p = 0.0369).Tumors with high intratumor heterogeneity also showed the tendency of having more total neoantigens.Conclusions:There is intratumor heterogeneity in dimensions of histology,mutation,transcriptome and immune microenvironment in HCC.In the branch evlution of HCC,the genetic mutation may be associated with relevant changes of gross morphology,histological architecture and immune cell infiltration.In AJCC Ⅰ stage HCC cohorts from TCGA,the group with high intratumor heterogeneity is associated with better disease specific survival rate and progression free survival rate.Finallly,tumors with high intratumor heterogeneity are associated with more immune cell infiltration and stronger cytolytic activity.It also shows the tendency of having more total neoantigens.Our results support the notion that genetic heterogeneity may induce stronger antitumor immune response.However,the results needs to be validated in large prospective patient cohorts.