A pilot study of ultra-deep targeted sequencing of plasma DNA identifies driver mutations in hepatocellular carcinoma



Cellular components of solid tumors including DNA are released into the bloodstream, but data on circulating-free DNA (cfDNA) in hepatocellular carcinoma (HCC) are still scarce. This study aimed at analyzing mutations in cfDNA and their correlation with tissue mutations in patients with HCC. We included 8 HCC patients treated with surgical resection for whom we collected paired tissue and plasma/serum samples. We analyzed 45 specimens, including multiregional tumor tissue sampling (n = 24), peripheral blood mononuclear cells (PMBC, n = 8), plasma (n = 8) and serum (n = 5). Ultra-deep sequencing (5500× coverage) of all exons was performed in a targeted panel of 58 genes, including frequent HCC driver genes and druggable mutations. Mutations detected in plasma included known HCC oncogenes and tumor suppressors (e.g., TERT promoter, TP53, and NTRK3) as well as a candidate druggable mutation (JAK1). This approach increased the detection rates previously reported for mutations in plasma of HCC patients. A thorough characterization of cis mutations found in plasma confirmed their tumoral origin, which provides definitive evidence of the release of HCC-derived DNA fragments into the bloodstream. This study demonstrates that ultra-deep sequencing of cfDNA is feasible and can confidently detect somatic mutations found in tissue; these data reinforce the role of plasma DNA as a promising minimally invasive tool to interrogate HCC genetics.

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The authors thank the office of Scientific Computing at the Icahn School of Medicine at Mount Sinai (ISMMS) for providing computational resources and staff expertize, as well as the ISMMS Tissue Biorepository for providing the samples. Sequencing was performed at the Genomics Core Facility at ISMMS.


IL is supported by a grant from the Swiss National Science Foundation, from Foundation Roberto & Gianna Gonella and Foundation SICPA. DD is supported by the Grant for Studies Broadening from the Spanish Association for the Study of the Liver (Asociación Española para el Estudio del Hígado, AEEH) and the Cancer Research Grant from Nuovo Soldati Foundation. AJC is supported by a Ruth L. Kirschstein NRSA Institutional Research Training Grant (T32 CA078207). JvF is supported by a grant from the German Research Foundation (FE 1746/1-1). SNM is supported by grants from the Brazilian National Council for Scientific and Technological Development, and “Associacao Piauiense de Combate ao Cancer”. JML is supported by grants from the European Commission (Heptromic, proposal number 259744; HEPCAR, proposal number 667273-2), the Samuel Waxman Cancer Research Foundation, the Spanish National Health Institute (J.M.L: SAF-2016-76390), the Asociación Española para el Estudio del Cáncer and the U.S. Department of Defense (CA150272P1). AV is supported by the U.S. Department of Defense (CA150272P3) and the Tisch Cancer Institute (Cancer Center Grant P30 CA196521). SLF is supported by NIH grant DK56621 and the U.S. Department of Defense (Contract No. W81XWH-16-1-0455). This study was funded by the American Association for the Study of Liver Diseases Foundation (AASLDF) Alan Hofmann Clinical and Translational Award to AV.

Author’s contributions

Study concept and design: IL, CVM, AV. Acquisition of data: IL, DD, AC, JvF, SNM, DS, AS, SW, MIF, SNM, MM, SNT, PT, CA, MS, AV. Analysis and interpretation of data: IL, CVM, AS, JvF, SNM, PT, SNT, SLF, JML, MS, AV. Drafting of the manuscript: IL, CVM, AV. Critical revision of the manuscript for important intellectual content: IL, CVM, DD, AC, JvF, AS, DS, SNM, SW, MIF, SNM, MM, PT, SNT, SLF, JML, CA, MS, AV

Author information

Author notes

  1. These authors contributed equally: Ismail Labgaa, Carlos Villacorta-Martin.


  1. Department of Medicine, Liver Cancer Program, Tisch Cancer Institute, Graduate School of Biomedical Sciences, Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, USA

    • Ismail Labgaa
    • , Carlos Villacorta-Martin
    • , Delia D’Avola
    • , Amanda J. Craig
    • , Johann von Felden
    • , Sebastiao N. Martins-Filho
    • , Daniela Sia
    • , Parissa Tabrizian
    • , Scott L. Friedman
    • , Josep M. Llovet
    • , Myron Schwartz
    •  & Augusto Villanueva
  2. Department of Visceral Surgery, Lausanne University Hospital CHUV, Lausanne, Switzerland

    • Ismail Labgaa
  3. Liver Unit and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Clinica Universidad de Navarra, Pamplona, Spain

    • Delia D’Avola
  4. I. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    • Johann von Felden
  5. Department of Pathology, University of Sao Paulo School of Medicine, Sao Paulo, Brazil

    • Sebastiao N. Martins-Filho
  6. Department of Pathology and Laboratory Medicine, University Health Network, University of Toronto, Toronto, ON, Canada

    • Sebastiao N. Martins-Filho
  7. Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, USA

    • Ashley Stueck
    • , Stephen C. Ward
    • , M. Isabel Fiel
    •  & Swan N. Thung
  8. Department of Pathology, Dalhousie University, Halifax, NS, Canada

    • Ashley Stueck
  9. Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, USA

    • Milind Mahajan
  10. Department of Surgery, Icahn School of Medicine at Mount Sinai, New York, USA

    • Parissa Tabrizian
    •  & Myron Schwartz
  11. Department of Medicine, Icahn School of Medicine at Mount Sinai, Division of Hematology and Medical Oncology, New York, USA

    • Celina Ang
    •  & Augusto Villanueva
  12. Liver Cancer Translational Research Laboratory, BCLC Group, IDIBAPS, CIBEREHD, Hospital Clinic, Universitat de Barcelona, Catalonia, Spain

    • Josep M. Llovet
  13. Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain

    • Josep M. Llovet


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Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Augusto Villanueva.

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