Abstract
Hepatocellular carcinoma (HCC) is a cancer of substantial morphologic, genetic and phenotypic diversity. Yet we do not understand the relationship between intratumor heterogeneity and the associated morphologic/histological characteristics of the tumor. Using single-cell whole-genome sequencing to profile 96 tumor cells (30-36 each) and 15 normal liver cells (5 each), collected from three male patients with HBV-associated HCC, we confirmed that copy number variations occur early in hepatocarcinogenesis but thereafter remain relatively stable throughout tumor progression. Importantly, we showed that specific HCCs can be of monoclonal or polyclonal origins. Tumors with confluent multinodular morphology are the typical polyclonal tumors and display the highest intratumor heterogeneity. In addition to mutational and copy number profiles, we dissected the clonal origins of HCC using HBV-derived foreign genomic markers. In monoclonal HCC, all the tumor single cells exhibit the same HBV integrations, indicating that HBV integration is an early driver event and remains extremely stable during tumor progression. In addition, our results indicated that both models of metastasis, late dissemination and early seeding, have a role in HCC progression. Notably, early intrahepatic spreading of the initiating clone leads to the formation of synchronous multifocal tumors. Meanwhile, we identified a potential driver gene ZNF717 in HCC, which exhibits a high frequency of mutation at both single-cell and population levels, as a tumor suppressor acting through regulating the IL-6/STAT3 pathway. These findings highlight multiple distinct tumor evolutionary mechanisms in HCC, which suggests the need for specific treatment strategies.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (81522036, 81572292 and 81372648 to QG; 81672956 and 81472413 to CL); Basic Research Project from Technology Commission of Shanghai Municipality (17JC1402200) and National Program for Special Support of Eminent Professionals and Science to QG; the Key Program (QYZDB-SSW-SMC036), External Cooperation Program (GJHZ201312) and the National Key Basic Research Program of China (2015CB856000) to XZ.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
Morphologic and histological features of three HCC used for SCG. (PDF 3362 kb)
Supplementary information, Figure S2
Depth and distribution of coverage for each sequencing library based on SCG (A) and population sequencing (B). (PDF 485 kb)
Supplementary information, Figure S3
Somatic Mutation Pattern Spectrums of each HCC based on SCG. (PDF 264 kb)
Supplementary information, Figure S4
Sanger sequencing validation of 11 HBV integration sites. (PDF 543 kb)
Supplementary information, Figure S5
The technical amplification and sequencing error rate of MALBAC data of the single cell samples. (PDF 234 kb)
Supplementary information, Figure S6
The Q20 and Q30 bases fraction of the single cell samples. (PDF 59 kb)
Supplementary information, Figure S7
Statistical significance of gain and loss regions in the SCGs of the three HCC patients. (PDF 450 kb)
Supplementary information, Figure S8
The mutation landscape and driver-gene prediction. (PDF 227 kb)
Supplementary information, Figure S9
ZNF717 knockdown promoted growth, migration, adhesion and invasion and inhibited apoptosis in HCC cells. (PDF 1502 kb)
Supplementary information, Figure S10
ZNF717 knockdown in HCC enhanced MMP2, CD44 and ITGA3 expression. (PDF 190 kb)
Supplementary information, Figure S11
Immunohistochemistry staining of phosphorylated STAT3 and its prognostic significance in HCC patients. (PDF 707 kb)
Supplementary information, Figure S12
ZNF717 suppresses the transcription of STAT3. (PDF 650 kb)
Supplementary information, Table S1
Clinicopathologic information of the 3 HCC patients. (PDF 74 kb)
Supplementary information, Table S2
HBV integration sites detected by SCG. (PDF 53 kb)
Supplementary information, Table S3
Common CNV identified by SCG in monoclonal tumors H-PT and H-CM. (XLS 44 kb)
Supplementary information, Table S4
Somatic point mutations detected in the three HCC by SCG. (XLS 246 kb)
Supplementary information, Table S5
Common SNVs identified by Larva in the non-coding region of monoclonal tumors H-PT and H-CM. (XLS 125 kb)
Supplementary information, Data S1
Materials and Methods (PDF 224 kb)
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Duan, M., Hao, J., Cui, S. et al. Diverse modes of clonal evolution in HBV-related hepatocellular carcinoma revealed by single-cell genome sequencing. Cell Res 28, 359–373 (2018). https://doi.org/10.1038/cr.2018.11
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DOI: https://doi.org/10.1038/cr.2018.11
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