Review Article | Published:

Liquid biopsies come of age: towards implementation of circulating tumour DNA

Nature Reviews Cancer volume 17, pages 223238 (2017) | Download Citation


Improvements in genomic and molecular methods are expanding the range of potential applications for circulating tumour DNA (ctDNA), both in a research setting and as a 'liquid biopsy' for cancer management. Proof-of-principle studies have demonstrated the translational potential of ctDNA for prognostication, molecular profiling and monitoring. The field is now in an exciting transitional period in which ctDNA analysis is beginning to be applied clinically, although there is still much to learn about the biology of cell-free DNA. This is an opportune time to appraise potential approaches to ctDNA analysis, and to consider their applications in personalized oncology and in cancer research.

Key points

  • Cell-free DNA (cfDNA) is released predominantly by cell death into the bloodstream, although active secretion may have a role. Since the discovery of fetal cfDNA in the maternal circulation, cfDNA analysis has been rapidly implemented in clinical practice for noninvasive prenatal testing.

  • Mutations were first detected in cfDNA more than two decades ago, and interest in circulating tumour DNA (ctDNA) as a noninvasive cancer diagnostic has increased considerably with the development of molecular methods that permit the sensitive detection and monitoring of multiple classes of mutation.

  • ctDNA may have utility at almost every stage of the management of patients with cancer, including diagnosis, minimally invasive molecular profiling, treatment monitoring, the detection of residual disease and the identification of resistance mutations. ctDNA analysis may be broadly considered as a tool both for quantitative analysis of disease burden and for genomic analysis.

  • The identification of ctDNA in individuals before a cancer diagnosis, and in presymptomatic individuals, suggests the possibility of ctDNA analysis as a tool for earlier detection and screening. Noninvasive cancer classification or subtyping has also emerged as a possibility, although for early detection, both technical and biological factors introduce challenges to the detection of mutant DNA in plasma and its interpretation.

  • Monitoring multiple mutations in parallel can enhance the sensitivity of ctDNA detection, can be used to assess the clonal evolution of patients' disease and may identify resistance mutations before clinical progression is observed.

  • ctDNA analysis is beginning to transition from the research setting into the clinic. The US Food and Drug Administration and the European Medicines Agency have approved ctDNA tests for specific indications in the absence of evaluable tumour tissue. Analysis of gene panels in plasma has now become available as a potential clinical tool. Large studies are under way to establish the overall performance and clinical utility of such assays when a tumour biopsy is not available for analysis.

  • Potential applications of ctDNA have been demonstrated by a number of proof-of-principle studies. Prospective clinical trials are beginning to assess the clinical utility of ctDNA analysis for molecular profiling and disease monitoring. The increasing acceptance of ctDNA is enabling the field to move from exploratory ctDNA studies towards clinical trials in which ctDNA guides decision-making.

  • In order to fully exploit the potential utility of liquid biopsies, it is essential that the biology of cfDNA and ctDNA is explored further. The mechanisms of release and degradation, and the factors that affect the representation of ctDNA in plasma, are poorly understood. The nature of ctDNA will be clarified through both large, well-annotated clinical studies and in vivo studies in which variables can be controlled.

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The authors would like to thank all members of the Rosenfeld laboratory, in particular C. G. Smith, D. Gale, K. M. Patel and W. N. Cooper for expert advice and proofreading of this manuscript. They would like to thank D. Lo (The Chinese University of Hong Kong) for providing the image used in Figure 3a. The authors would also like to acknowledge the support of the University of Cambridge (UK), Cancer Research UK (grant numbers A11906, A20240 and A15601; to N.R. and J.D.B.), the European Research Council (ERC) under the European Union's Seventh Framework Programme (FP/2007-2013) ERC Grant Agreement number 337905 (to N.R.), Hutchison Whampoa Limited (Hong Kong; to N.R.), AstraZeneca (UK; to R.B. and S.P.), the Cambridge Experimental Cancer Medicine Centre (to R.B. and S.P.), and the National Institute for Health Research Biomedical Research Centre (UK; to R.B. and S.P.). J.G.-C. acknowledges clinical fellowship support from the Spanish Society of Medical Oncology (SEOM).

Author information

Author notes

    • Richard Baird
    •  & Nitzan Rosenfeld

    These authors contributed equally to this work.


  1. Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.

    • Jonathan C. M. Wan
    • , Charles Massie
    • , Florent Mouliere
    • , James D. Brenton
    • , Carlos Caldas
    •  & Nitzan Rosenfeld
  2. Cancer Research UK Cambridge Centre, Cambridge CB2 0RE, UK.

    • Jonathan C. M. Wan
    • , Charles Massie
    • , Florent Mouliere
    • , James D. Brenton
    • , Carlos Caldas
    • , Simon Pacey
    • , Richard Baird
    •  & Nitzan Rosenfeld
  3. Clinical Trials Unit, Clinic Institute of Haematological and Oncological Diseases, Hospital Clinic de Barcelona, IDIBAPs, Carrer de Villarroel, 170 Barcelona 08036, Spain.

    • Javier Garcia-Corbacho
  4. Department of Oncology, University of Cambridge Hutchison–MRC Research Centre, Box 197, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK.

    • Carlos Caldas
    • , Simon Pacey
    •  & Richard Baird


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Competing interests

N.R. is the Chief Scientific Officer of Inivata (Cambridge, UK, and Research Triangle Park, North Carolina, USA). N.R. and J.D.B. are co-founders and shareholders of Inivata. N.R. and F.M. are co-inventors of patent applications that describe methods for the analysis of DNA fragments and applications of circulating tumour DNA.

Corresponding author

Correspondence to Nitzan Rosenfeld.


Liquid biopsy

Analysis of tumour material (for example, cells or nucleic acids) obtained in a minimally invasive or noninvasive manner through the sampling of blood or other body fluids.

Digital PCR

(dPCR). An assay in which many microlitre- or nanolitre-scale PCR reactions are run in parallel within physically separated reaction chambers or as droplets in an emulsion (droplet dPCR (ddPCR)). By partitioning molecules into hundreds or up to millions of reactions, rare mutant molecules can be accurately identified and quantified.

Mutant allele fraction

The proportion of mutant DNA fragments at a given locus.

Hybrid-capture sequencing

DNA sequencing of kilobases to megabases of the genome, in which the DNA to be sequenced is selected using complementary oligonucleotide baits that hybridize to the target DNA. The DNA is then captured in solution, commonly through binding to magnetic beads.

Targeted sequencing

Massively parallel (next-generation) sequencing that uses methods such as PCR amplification or hybrid capture to focus on a subset of the genome, which can range from few genes or mutation loci to large proportions of the genome, such as the entire exome. Smaller panels yield a higher sequencing depth at lower costs than do larger panels.

Molecular barcoding

The addition of unique molecular sequences to each molecule when creating a sequencing library, so that reads originating from the same molecule can be identified and the consensus taken, correcting for some PCR or sequencing errors.

Limit of detection

The threshold below which mutations cannot be confidently discriminated from background noise; for sequencing-based approaches, this is often determined by technical artefacts such as PCR or sequencing errors.

Mutant allele concentration

The number of mutant DNA fragments at a given locus per unit volume.

Stem mutations

Mutations that occur early in a cancer's development and are present in all cells.

Private mutations

Mutations that are present only in a specific region of a tumour, or in a subset of cells, owing to intratumour heterogeneity.

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