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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  1. 1.

    Crowley E, Di Nicolantonio F, Loupakis F, Bardelli A. Liquid biopsy: monitoring cancer-genetics in the blood. Nat Rev Clin Oncol. 2013;10:472–84.

  2. 2.

    Dawson SJ, Tsui DW, Murtaza M, Biggs H, Rueda OM, Chin SF, et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med. 2013;368:1199–209.

  3. 3.

    Murtaza M, Dawson SJ, Tsui DW, Gale D, Forshew T, Piskorz AM, et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature. 2013;497:108–12.

  4. 4.

    Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014;6:224ra224.

  5. 5.

    Mandel P, Metais P. Les acides nucléiques du plasma sanguin chez lahomme. C R Seances Soc Biol Fil. 1948;142:241–3.

  6. 6.

    Jiang P, Chan CW, Chan KC, Cheng SH, Wong J, Wong VW, et al. Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients. Proc Natl Acad Sci USA. 2015;112:E1317–25.

  7. 7.

    Lin XJ, Chong Y, Guo ZW, Xie C, Yang XJ, Zhang Q, et al. A serum microRNA classifier for early detection of hepatocellular carcinoma: a multicentre, retrospective, longitudinal biomarker identification study with a nested case-control study. Lancet Oncol. 2015;16:804–15.

  8. 8.

    Russo M, Siravegna G, Blaszkowsky LS, Corti G, Crisafulli G, Ahronian LG, et al. Tumor heterogeneity and lesion-specific response to targeted therapy in colorectal cancer. Cancer Discov. 2016;6:147–53.

  9. 9.

    Tie J, Wang Y, Tomasetti C, Li L, Springer S, Kinde I, et al. Circulating tumor DNA analysis detects minimal residual disease and predicts recurrence in patients with stage II colon cancer. Sci Transl Med. 2016;8:346ra392.

  10. 10.

    Maemondo M, Inoue A, Kobayashi K, Sugawara S, Oizumi S, Isobe H, et al. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med. 2010;362:2380–8.

  11. 11.

    FDA approves first blood test to detect gene mutation associated with non-small cell lung cancer [news release]. Silver Spring, MD: U.S. Food and Drug Administration. http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm504488.htm. Accessed June 1, 2016.

  12. 12.

    Liao W, Yang H, Xu H, Wang Y, Ge P, Ren J, et al. Noninvasive detection of tumor-associated mutations from circulating cell-free DNA in hepatocellular carcinoma patients by targeted deep sequencing. Oncotarget. 2016;7:40481–90.

  13. 13.

    Huang A, Zhao X, Yang XR, Li FQ, Zhou XL, Wu K, et al. Circumventing intratumoral heterogeneity to identify potential therapeutic targets in hepatocellular carcinoma. J Hepatol. 2017;67:293–301.

  14. 14.

    Cai ZX, Chen G, Zeng YY, Dong XQ, Lin MJ, Huang XH, et al. Circulating tumor DNA profiling reveals clonal evolution and real-time disease progression in advanced hepatocellular carcinoma. Int J Cancer. 2017;141:977–85.

  15. 15.

    Llovet JM, Zucman-Rossi J, Pikarsky E, Sangro B, Schwartz M, Sherman M, et al. Hepatocellular carcinoma. Nat Rev Dis Prim. 2016;2:16018.

  16. 16.

    European Association For The Study Of The L, European Organisation For R, Treatment Of C. EASL-EORTC clinical practice guidelines: management of hepatocellular carcinoma. J Hepatol. 2012;56:908–43.

  17. 17.

    Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem. 2011;83:8604–10.

  18. 18.

    Kan Z, Zheng H, Liu X, Li S, Barber TD, Gong Z, et al. Whole-genome sequencing identifies recurrent mutations in hepatocellular carcinoma. Genome Res. 2013;23:1422–33.

  19. 19.

    De Mattos-Arruda L, Weigelt B, Cortes J, Won HH, Ng CK, Nuciforo P, et al. Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle. Ann Oncol. 2014;25:1729–35.

  20. 20.

    Zhai W, Lim TK, Zhang T, Phang ST, Tiang Z, Guan P, et al. The spatial organization of intra-tumour heterogeneity and evolutionary trajectories of metastases in hepatocellular carcinoma. Nat Commun. 2017;8:4565.

  21. 21.

    Norton ME, Jacobsson B, Swamy GK, Laurent LC, Ranzini AC, Brar H, et al. Cell-free DNA analysis for noninvasive examination of trisomy. N Engl J Med. 2015;372:1589–97.

  22. 22.

    Malapelle U, Mayo de-Las-Casas C, Rocco D, Garzon M, Pisapia P, Jordana-Ariza N, et al. Development of a gene panel for next-generation sequencing of clinically relevant mutations in cell-free DNA from cancer patients. Br J Cancer. 2017;116:802–10.

  23. 23.

    Zill OA, Greene C, Sebisanovic D, Siew LM, Leng J, Vu M, et al. Cell-free DNA next-generation sequencing in pancreatobiliary carcinomas. Cancer Discov. 2015;5:1040–8.

  24. 24.

    Huang A, Zhang X, Zhou SL, Cao Y, Huang XW, Fan J, et al. Detecting circulating tumor DNA in hepatocellular carcinoma patients using droplet digital PCR is feasible and reflects intratumoral heterogeneity. J Cancer. 2016;7:1907–14.

  25. 25.

    Ono A. Circulating tumor DNA analysis for liver cancers and its usefulness as a liquid biopsy. Cell Mol Gastroenterol Hepatol. 2015;1:516–34.

  26. 26.

    Labgaa I, Villanueva A. Liquid biopsy in liver cancer. Discov Med. 2015;19:263–73.

  27. 27.

    Newman AM, Bratman SV, To J, Wynne JF, Eclov NC, Modlin LA, et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat Med. 2014;20:548–54.

  28. 28.

    Schulze K, Imbeaud S, Letouze E, Alexandrov LB, Calderaro J, Rebouissou S, et al. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nat Genet. 2015;47:505–11.

  29. 29.

    Izumchenko E, Chang X, Brait M, Fertig E, Kagohara LT, Bedi A, et al. Targeted sequencing reveals clonal genetic changes in the progression of early lung neoplasms and paired circulating DNA. Nat Commun. 2015;6:8258.

  30. 30.

    Newman AM, Lovejoy AF, Klass DM, Kurtz DM, Chabon JJ, Scherer F, et al. Integrated digital error suppression for improved detection of circulating tumor DNA. Nat Biotechnol. 2016;34:547–55.

  31. 31.

    Abbosh C, Birkbak NJ, Wilson GA, Jamal-Hanjani M, Constantin T, Salari R, et al. Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature. 2017;545:446–51.

  32. 32.

    Siravegna G, Mussolin B, Buscarino M, Corti G, Cassingena A, Crisafulli G, et al. Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients. Nat Med. 2015;21:795–801.

  33. 33.

    Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011;364:2507–16.

  34. 34.

    Zehir A, Benayed R, Shah RH, Syed A, Middha S, Kim HR, et al. Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat Med. 2017;23:703–13.

  35. 35.

    Totoki Y, Tatsuno K, Covington KR, Ueda H, Creighton CJ, Kato M, et al. Trans-ancestry mutational landscape of hepatocellular carcinoma genomes. Nat Genet. 2014;46:1267–73.

  36. 36.

    Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25:1754–60.

  37. 37.

    McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20:1297–303.

  38. 38.

    Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, Levy-Moonshine A, et al. From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr Protoc Bioinform. 2013;43:11 10 11–33.

  39. 39.

    Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, Lin L, et al. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res. 2012;22:568–76.

  40. 40.

    Chang MT, Asthana S, Gao SP, Lee BH, Chapman JS, Kandoth C, et al. Identifying recurrent mutations in cancer reveals widespread lineage diversity and mutational specificity. Nat Biotechnol. 2016;34:155–63.

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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

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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.

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Correspondence to Augusto Villanueva.

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