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

Journal name:
Nature Reviews Cancer
Year published:
Published online


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.

At a glance


  1. Applications of circulating tumour DNA analysis during the course of disease management.
    Figure 1: Applications of circulating tumour DNA analysis during the course of disease management.

    a | A schematic time course for a hypothetical patient who undergoes surgery (or other initial treatment), has a disease relapse and then receives systemic therapy. The potential applications of liquid biopsies during this patient's care are indicated. The patient starts with one single disease focus, but multiple metastases and distinct clones (depicted in different colours) emerge following treatment. b | The information extracted from circulating tumour DNA (ctDNA) may be classified, broadly, as quantitative information (that is, relating to tumour burden) or genomic information. Quantification of ctDNA at a single time point may allow disease staging and prognostication, and genomic analysis can inform the selection of targeted therapies. Therefore, longitudinal analysis allows the quantitative tracking of tumour burden to monitor treatment response, for example, and by comparing genomic profiles over time, clonal evolution may be monitored.

  2. Origins and range of alterations in cell-free DNA.
    Figure 2: Origins and range of alterations in cell-free DNA.

    Cells release cell-free DNA (cfDNA) through a combination of apoptosis, necrosis and secretion. cfDNA can arise from cancerous cells but also from cells in the tumour microenvironment, immune cells or other body organs. In the bloodstream, cfDNA may exist as either free or associated with extracellular vesicles such as exosomes2. Multiple classes of genetic and epigenetic alterations can be found in cfDNA. Adapted with permission from Ref. 215, Macmillan Publishers Ltd.

  3. Current and future paradigms for the sensitive detection of circulating tumour DNA.
    Figure 3: Current and future paradigms for the sensitive detection of circulating tumour DNA.

    a | The analysis of cell-free (cfDNA) can range from the interrogation of individual loci to analysis of the whole genome (Table 1). Off-the-shelf digital PCR (dPCR) assays can achieve high sensitivity with a simple workflow but are limited by a low multiplexing capability. Targeted sequencing can allow the interrogation of multiple loci with high sensitivity through the use of methods that suppress background noise242.b | In molecular barcoding, unique molecular sequences are added to each molecule during library preparation so that sequencing reads originating from the same starting molecule can be identified. By comparing all reads from the same molecule, a single consensus sequence can be taken, thus suppressing errors arising from PCR or sequencing. c | To improve the sensitivity of the analysis — for example, for disease diagnosis or detection of minimal residual disease — other body fluids may be considered in combination with, or instead of, plasma. Sampling of body fluids or cytological specimens proximal to the tumour site may yield a higher concentration of DNA of tumour origin than is found in plasma. d | Circulating tumour DNA (ctDNA) has been shown to be shorter than cfDNA4, 80, 81, 84. Thus, experimental or in silico selection of shorter fragments may enrich for sequences of cancer origin81, and can improve sensitivity for samples with low proportions of ctDNA. In part a, the targeted sequencing image is adapted from Ref. 34 (Forshew, T. et al. Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci. Transl Med. 4, 136ra68 (2012)). Reproduced with permission from the AAAS. The whole-genome sequencing image is courtesy of Dennis Lo (The Chinese University of Hong Kong) and is based on data published in Ref. 37.

  4. Leveraging multiple mutations to detect low-burden disease and overcome sampling noise.
    Figure 4: Leveraging multiple mutations to detect low-burden disease and overcome sampling noise.

    Even with a perfectly sensitive assay, the probability of detecting circulating tumour DNA (ctDNA) decreases as ctDNA concentration declines, as any single mutation of interest may not be present in a given volume of sample. At low ctDNA concentrations, some mutations will be detected while others are missed owing to sampling error. Sampling multiple pre-specified mutations in each reaction may improve the detection of low levels of ctDNA, as every target provides an independent opportunity to test for the presence of a mutant molecule in the set of DNA molecules at that locus34, 114. Sensitivity can be further improved by analysing multiple replicates that each contain few molecules, so that the mutant allele — where present in a reaction — will constitute a large proportion of the DNA template116. The dashed boxes below the graph show hypothetical examples of sets of molecules that may be captured by each replicate in the analysis of a sample.

  5. Adaptive or reactive treatment paradigms using liquid biopsies.
    Figure 5: Adaptive or reactive treatment paradigms using liquid biopsies.

    a | During systemic anticancer therapy, serial liquid biopsies may identify biochemical response or progression. If progression is identified, the clinician may be able to switch therapy or select a therapy to target arising clones that carry additional mutations that were identified by the analysis. b | This adaptive or reactive monitoring and treatment may continue as a loop, which would be facilitated by a fast turnaround time for circulating tumour DNA (ctDNA) analysis, for example through the use of point-of-care diagnostics. The time frames for this analysis can vary between hours and months; a time frame of hours could allow the analysis of early kinetics in response to therapy. EGFR, epidermal growth factor receptor; NSCLC, non-small-cell lung cancer; PIK3CAMUT, mutant PI3K catalytic subunit-α.


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Author information

  1. These authors contributed equally to this work.

    • Richard Baird &
    • Nitzan Rosenfeld


  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

Competing interests statement

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:

Author details

  • Jonathan C. M. Wan

    Jonathan C. M. Wan is an M.D.–Ph.D. student at Trinity College, University of Cambridge, UK. He completed his preclinical medical studies at King's College London, UK. At present, he is carrying out his Ph.D. in the group of Nitzan Rosenfeld at the Cancer Research UK Cambridge Institute, University of Cambridge (2015–18), where he is working on developing individualized circulating tumour DNA assays to monitor patients with melanoma with high sensitivity.

  • Charles Massie

    Charles Massie trained as a biochemist and then obtained a Ph.D. in Oncology at the University of Cambridge, UK. With a particular interest in the biology and early detection of prostate cancer, his research combines computational approaches, cancer genomics and high-sensitivity approaches for the analysis of tumour DNA in peripheral fluids.

  • Javier Garcia-Corbacho

    Javier García-Corbacho completed his medical oncology training at Hospital Universitario Reina Sofía (Córdoba, Spain) and his research training at the University of Cambridge, UK. He is currently the Head of the Clinical Trials Unit of the Clinic Institute of Haematological and Oncological Diseases (ICMHO, Barcelona, Spain), where he leads the Early Phase Clinical Trials Unit. His translational research interests include gene profiling and circulating tumour DNA.

  • Florent Mouliere

    Florent Mouliere is a postdoctoral research associate at the Cancer Research UK Cambridge Institute, University of Cambridge, UK. He has an interest in tailoring new sequencing technologies to the study of the biology of circulating nucleic acids and to improve the sensitivity of liquid biopsy in difficult pathological contexts (for example, brain tumours, early response kinetics and early-stage cancers). He received his Ph.D. from the University of Montpellier, France.

  • James D. Brenton

    James D. Brenton is a medical oncologist and senior group leader at the Cancer Research UK Cambridge Institute, University of Cambridge, UK, where he leads the Functional Genomics of Ovarian Cancer Laboratory. His research focuses on identifying predictive genomic biomarkers for therapy in ovarian cancer and elucidating mechanisms of drug resistance.

  • Carlos Caldas

    Carlos Caldas is the Professor of Cancer Medicine at the University of Cambridge, UK; Head of the Breast Cancer Functional Genomics Laboratory at the Cancer Research UK Cambridge Institute; and Director of the Breast Cancer Programme at Cancer Research UK Cambridge Centre. His laboratory has published extensively on breast cancer genomics and he is a pioneer of circulating tumour DNA applications.

  • Simon Pacey

    Simon Pacey is an academic consultant in medical oncology who specializes in early-phase and phase I clinical trials. His site-specific interest is prostate malignancy. His research interests include leading a portfolio of early-phase oncology trials and translational work to study the effects of drugs in men with localized, hormone-sensitive prostate cancer.

  • Richard Baird

    Richard Baird is an academic medical oncologist at the Cancer Research UK Cambridge Centre. He runs a portfolio of early-phase clinical trials that are focused on novel breast cancer therapeutics and biomarker-directed treatment strategies. He is the chief investigator on three investigator-initiated trials and is UK Academic Lead for the Association of Cancer Physicians.

  • Nitzan Rosenfeld

    Nitzan Rosenfeld trained in physics, systems biology and biotechnology. Since 2009, he has been leading a translational research group at the Cancer Research UK Cambridge Institute, University of Cambridge, UK, that is devoted to circulating tumour DNA (ctDNA)-based molecular diagnostics, establishing techniques for ctDNA analysis and the applications of ctDNA in oncology. He is co-founder and Chief Scientific Officer of Inivata (Cambridge, UK, and Research Triangle Park, North Carolina, USA), which applies ctDNA sequencing for precision medicine.

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