Whole-exome Sequencing Reveals The Origin And Evolution Of Combined Hepatocellular Cholangiocarcinoma

BACKGROUND:Hepato-Cholangiocarcinoma(CHC)is a rare subtype of primary liver cancer with clinicopathological features of both hepatocellular carcinoma(HCC)and intrahepatic cholangiocarcinoma(iCCA).However,molecular mechanisms underlying the co-existence of HCC and iCCA components in a single tumor remain elusive.Herein,we aim to investigate the relationship of these two tumor components in CHC.METHODS:Seventy-five liver cancer patients including 15 with CHC,32 with HCC,and 28 with iCCA were enrolled in this study.Immunohistochemical staining(IHC)was performed for all samples using hepatocytic markers(Hep and GPC3)and biliary epithelial markers(CK7 and CK19).Seven CHC samples were selected for laser microdissection isolating HCC,iCCA,and the adjacent noncancerous liver tissues.DNA extracted from HCC and iCCA were subjected for whole exome sequencing(WES).Nonsynonymous mutations,somatic copy number variations(CNVs),driver mutations,significantly mutated genes,as well as hepatitis B virus(HBV)integrations were analyzed.To further study the progenitor cell features of CHC,IHC staining with stem cell markers(EpCAM and c-kit)was conducted.In addition,we performed a Kaplan-Meier survival analysis to evaluate the correlation between protein levels of such stem cell markers and mortality.RESULTS:CHC was further confirmed by IHC staining.As to WES results,we defined somatic mutations shared by HCC and iCCA as ubiquitous mutations,whereas mutations distinct in each component as private mutations.We found that CHC samples contain substantial private somatic single nucleotide polymorphism(SNV)(ranging from 33.1%to 86.4%)as well as private somatic copy number variants(CNVs)(ranging from 79.3%to 97.3%),indicative of intratumor heterogeneity(ITH)of CHC.However,large amount of ubiquitous nonsynonymous mutations and CNVs were overlapped in HCC and iCCA samples,suggesting the monoclonal origin of CHC.Clonal analyses also supported the similar conclusion.Mutated genes identified herein e.g.VCAN,ACVR2A and FCGBP that related to the stem cell status and cell differentiation are speculated to contribute to distinct differentiation of HCC and IHC within CHC.Moreover,with regard to the progenitor cell feature analysis,EpCAM was positive in 80%of all CHC samples.Among HCC patients,EpCAM positivity in CK19(?)vs.CK19(-)samples is 66.7%and 17.2%,respectively.And 71.4%of iCCA samples had positive EpCAM staining.Kaplan-Meier curve showed that EpCAM expression was associated with poor prognosis of patients with liver cancer.CONCLUSIONS:Exome sequencing analyses are suggestive of the monoclonal origin of CHC,which may promote the molecule classification of primary liver cancer on the basis of cell origin.In addition,the substantial intratumor heterogeneity noted in CHC urges multiregional sequencing analysis to find the common driver mutations that playing more important role in carcinogenesis,thus make target drugs selection more precision and effective.