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. 2018 Mar 1;9(1):894.
doi: 10.1038/s41467-018-03276-y.

Whole-exome Sequencing Reveals the Origin and Evolution of Hepato-Cholangiocarcinoma

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Free PMC article

Whole-exome Sequencing Reveals the Origin and Evolution of Hepato-Cholangiocarcinoma

Anqiang Wang et al. Nat Commun. .
Free PMC article

Abstract

Hepatocellular-cholangiocarcinoma (H-ChC) is a rare subtype of liver cancer with clinicopathological features of both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). To date, molecular mechanisms underlying the co-existence of HCC and iCCA components in a single tumor remain elusive. Here, we show that H-ChC samples contain substantial private mutations from WES analyses, ranging from 33.1 to 86.4%, indicative of substantive intratumor heterogeneity (ITH). However, on the other hand, numerous ubiquitous mutations shared by HCC and iCCA suggest the monoclonal origin of H-ChC. Mutated genes identified herein, e.g., VCAN, ACVR2A, and FCGBP, are speculated to contribute to distinct differentiation of HCC and iCCA within H-ChC. Moreover, immunohistochemistry demonstrates that EpCAM is highly expressed in 80% of H-ChC, implying the stemness of such liver cancer. In summary, our data highlight the monoclonal origin and stemness of H-ChC, as well as substantial intratumoral heterogeneity.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Flowchart of patient selection and experimental procedure. A total of 32 hepatocelluar carcinoma (H-ChC), 28 cholangiocarcinoma (iCCA), and 15 hepato-cholangiocarcinoma (H-ChC) were selected for immunohistochemistry. The blackline in the pathological picture of H-ChC separate HCC component from iCCA component in H-ChC. Finally, seven H-ChC patients were included for microdissection and DNA extraction. Then, we conducted whole-exome sequencing and exome data analysis
Fig. 2
Fig. 2
Immunohistochemical profiles of liver cancer. a The tumor cells show immunoreactivity for Heptocyte (Hep), GPC-3, CK7, and CK19 in H-ChC (P6). Immunocreativity for Hep and GPC-3 are observed in HCC. Tumor cells are positive for CK7, CK19 in iCCA. b The columns show the expression level of Hep/GPC3 and CK7/19 in H-ChC, HCC, and iCCA. The scale bar represents 1 mm
Fig. 3
Fig. 3
Distribution of nonsynonymous SNVs. a The cartoons of liver show the tumor sites in patients. b Venn diagrams show the relationship of nonsynonymous somatic mutations between H-ChC component (red circle) and iCCA component (green circle) in every H-ChC patient. The different number represents the mumber of nonsynonymous somatic mutations for corresponding samples and the overlapped regions are ubiquitous nonsynonymous somatic mutations between two samples of same H-ChC patients
Fig. 4
Fig. 4
Mutation spectra and mutation signatures among H-ChC samples. a The upper column depicts mutation spectrum within all tumor samples. Red represents mutation type C>G/G>C, blue T>G/A>C, green T>A/A>T, purple T>C/A>G, orange C>A/G>T, and yellow C>T/G>A. Y axis indicates fraction of matations. The below column shows mutation signatures of all tumor samples. b The upper chart depicts the distribution of somatic copy number variation (CNV) of patient 3 (P3). Red is for CNV gain, green for normal CNV, and blue for CNV loss. The below chart depicts the distribution of B allele frequency (BAF). Orange represents consistent distribution of allele and blue indicates loss of heterozygosity (LOH). Heat maps show the distribution of CNV for all included H-ChC patients. Red is for CNV gain and blue for CNV loss. c Circos plot depicts the relationship between HCC and iCCA components of P5 on the terms of somatic nucleotide variant (SNV), somatic insertions and deletions, somatic copy number variation (CNV), and HBV integration. The first and second circles represent CNV for iCCA and HCC. Red indicates CNV gain, green normal CNV, and blue CNV loss. The third and fourth circles represent SNV and indel for iCCA and HCC. Green dot indicates SNV and indel. Green curve indicates HBV integration sites for iCCA and red curve for HCC. d Significantly mutated genes (SMG) landscape shows the distribution of some SMGs between samples in H-ChC. The column on top shows the mutational rate of every sample. Heat map shows the SMGs and mutation type including missense mutation (red block), nonsense mutation (light blue block), and shift indel (dark blue block). H1 and C1 represent the HCC and iCCA components of patient 1 (P1). Similar label was used for other patients. The column on the left stands for the percent of mutations for SMGs. The picture on the right shows the P value for SMGs
Fig. 5
Fig. 5
Missense mutations and cancer cell fraction comparisons within each H-ChC. a Heatplot with different bars represents various distributions of missense mutations including somatic SNVs and Indels within H-ChC. Fraction of ubiquitous nonsynonymous somatic mutations (trunk) (blue bar) and unique nonsynonymous somatic mutations (branch) (green bar for HCC and pink bar for iCCA) reveal the relationship of two tumor samples within a single H-ChC. Part of driver mutations was marked on trunk and branch of evolutionary trees. b Two-dimensional scatter plots show the cancer cell fraction (CCF) of the mutations in HCC and iCCA components of tumors. Different clusters were calculated from each H-ChC sample. Clusters off the axes indicate mutations in both of tumor components. Clusters on the axes reveal mutations in either HCC or iCCA components. c The table shows missense mutations in different tumor components
Fig. 6
Fig. 6
Expression of stem cell markers and survival analysis. a The tumor cells show immunoreactivity for EpCAM in H-ChC. b The columns show the expression level of EpCAM in H-ChC, HCC, and iCCA. c The survival curve shows the relationship between EpCAM expression and suivival. The scale bar represents 1 mm

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