1
Liquid biopsies come of age: towards implementation of
circulating tumour DNA
Jonathan C. M. Wan
1,2
, Charles Massie
1,2
, Javier Garcia-Corbacho
3
, Florent
Mouliere
1,2
, James D. Brenton
1,2
, Carlos Caldas
1,2,4
, Simon Pacey
2,4
, Richard
Baird
2,4
*, Nitzan Rosenfeld
1,2
*
1
Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge
2
Cancer Research UK Cambridge Centre, Cambridge CB2 0RE
3
Clinical Trials Unit, Clínic Institute of Haematological & Oncological Diseases, Hospital Clínic
de Barcelona, IDIBAPs, Carrer de Villarroel, 170 Barcelona 08036
4
Department of Oncology, University of Cambridge
*RB and NR contributed equally.
Please send correspondence to NR at nitzan.rosenfeld@cruk.cam.ac.uk.
Key points
• cfDNA is released predominantly by cell death into the bloodstream, though
active secretion may play a role. Since the discovery of fetal cfDNA in the
maternal circulation, cfDNA analysis has been rapidly implemented in clinical
practice for non-invasive prenatal testing.
• Mutations were first detected in cfDNA over two decades ago, and interest in
ctDNA as a non-invasive cancer diagnostic has increased dramatically 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 cancer patient management,
including: diagnosis, minimally invasive molecular profiling, treatment
monitoring, detection of residual disease, and identification of resistance
mutations. ctDNA analysis may be broadly considered as a tool for both
quantitative analysis of disease burden and for genomic analysis.
• The identification of ctDNA in individuals prior to a cancer diagnosis, and in
pre-symptomatic individuals, suggests the possibility of ctDNA analysis as a
2
tool for earlier diagnosis or screening. Non-invasive cancer classification or
sub-typing has also emerged as a possibility, though 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 sensitivity for ctDNA
detection, can be used to assess 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. Larger studies are underway 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. Increasing acceptance of ctDNA is enabling the field to move from
exploratory ctDNA studies towards clinical trials where ctDNA is guiding
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. 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 through in vivo studies, where
variables may be controlled.
3
Preface
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 at an exciting transitional period where
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 for ctDNA analysis, and to consider their applications in
personalised oncology and in cancer research.
The presence of fragments of cell-free nucleic acids in human blood was first
described in 1948 by Mandel and Métais
1
. The origins and characteristics of cell-free
DNA (cfDNA) were studied intermittently in subsequent decades
2
. In healthy
individuals, cfDNA concentration tends to range between 1-10ng/millilitre (ml) in
plasma
3,4
. Raised cfDNA levels were first reported in the serum of cancer patients in
1977
5
; cfDNA concentration can also be raised by other physiological conditions or
clinical scenarios, such as acute trauma
6
, cerebral infarction
7
, exercise
8
,
transplantation
9
, and infection
10
. Furthermore, the identification of fetal DNA
sequences in maternal plasma by Dennis Lo and colleagues in 1997
11
has led to
multiple applications of cfDNA in prenatal medicine including sex determination
12
,
identification of monogenic disorders
13
, and non-invasive prenatal testing (NIPT) for
aneuploidies such as Down’s Syndrome (trisomy 21). NIPT was first demonstrated in
2007 by Lo et al.
14
and has moved rapidly into widespread clinical use
15,16
.
In 1989, Stroun, Anker et al. identified that at least some cfDNA in the plasma of
cancer patients originates from cancer cells
2,17
. In 1991, Vogelstein, Sidransky and
colleagues showed that DNA from urinary sediments (cell pellets) from patients with
invasive bladder cancer carried mutations in TP53, setting the stage for the use of
genomic analysis methods in liquid biopsy applications
18
. KRAS mutations were soon
found in stool or sputum that matched mutations from colorectal
19
, pancreatic
20
or
lung
21,22
cancers. In 1994, mutated KRAS sequences were first reported to be
4
detected in plasma cfDNA of patients with pancreatic cancer using polymerase chain
reaction (PCR) with allele-specific primers
23
. For each patient, the KRAS mutation
found in the plasma was identical to that found in the patient’s tumour, thereby
confirming that the mutant DNA fragments in plasma were of tumour origin.
Mutations in cfDNA are highly specific markers for cancer, which gave rise to the
term circulating tumour DNA (ctDNA).
In the following decades, ctDNA was explored as a prognostic or predictive
marker
24,25
and for cancer detection
26
. Such studies confirmed the potential of ctDNA,
though the levels of ctDNA in different clinical contexts were not yet accurately
defined. These studies nonetheless could demonstrate potential clinical applications,
for example detection of KRAS mutations in plasma as a potential prognostic factor
in colorectal cancer
27
. The introduction of a digital PCR (dPCR) method in 1999 by
Vogelstein and Kinzler enabled the accurate identification and absolute quantification
of rare mutant fragments
28
. A modification of this technique using beads in
emulsions
29
and flow cytometry allowed the quantification of the mutant allele fraction
of cancer mutations in the plasma of patients with different stages of colorectal
cancer
30
. Diehl, Diaz et al. then showed in 2008 that ctDNA is a highly specific
marker of tumour dynamics, and may be able to indicate residual disease
31
. In
parallel, allele-specific PCR and other methods were devised and tested for their
ability to identify epidermal growth factor receptor (EGFR) mutations in serum or
plasma of lung cancer patients
32
, following the elucidation of the role of such
mutations in predicting response to treatment with molecularly targeted
inhibitors
25,33,34
.
The development of next generation sequencing-based technologies has facilitated
the interrogation of the genome at a broader scale. In 2012, deep sequencing of
multiple genes in cfDNA was demonstrated using panels of tagged amplicons, which
allowed the identification of mutations directly in the plasma of cancer patients, and
monitoring of multiple tumour-specific mutations in a single assay
35
. This method was
subsequently applied to monitor ctDNA in a cohort of patients with metastatic breast
cancer
36
. Shortly thereafter, whole-genome sequencing (WGS) of plasma cfDNA was
5
first shown to identify tumour-derived chromosomal aberrations
37
, focal
amplifications
38
and gene rearrangements
39
, and hybrid-capture sequencing was
introduced as a non-invasive method to analyse the evolving genomic profile of
mutations in cancer across the entire exome
40
.
There is a clear clinical need for novel diagnostic and molecular tools in oncology
(Box 1). Conventional sampling methods such as needle biopsies are subject to
procedural complications in up to one in six biopsies
41
, difficulty in obtaining sufficient
material of adequate quality for genomic profiling (reported failure rates range from
<10% to >30% of cases)
42,43
, and sampling biases arising from genetic
heterogeneity
44–48
. Detection and monitoring of disease often relies on body fluid-
based markers that often lack specificity
49
, and imaging which exposes patients to
ionising radiation
50
and has limited resolution (in both time and space). Recent
advances in ctDNA research highlight the potential applications of liquid biopsies at
each stage of patient management (Fig. 1a). These potential applications primarily
arise from two types of information obtainable through ctDNA analysis: quantification
of disease burden, and genomic analysis of cancer (Fig. 1b). These may be
combined and/or leveraged through serial sampling in order to monitor disease
burden and clonal evolution.
The increasing availability and reliability of techniques for PCR and high-throughput
sequencing are facilitating novel high-sensitivity applications, the generation of large
clinical datasets, and a better understanding of the origin of both cfDNA and ctDNA.
This Review will highlight and explore recent advances in the field and the
implications for oncology.
cfDNA and ctDNA biology
Characteristics of cfDNA and ctDNA
cfDNA is thought to be released from cells mostly through apoptosis and necrosis,
and possibly also active secretion
2,51–54
. Outside of the blood circulation, cfDNA has
been detected a variety of body fluids including urine
55–58,59
, cerebrospinal fluid
(CSF)
60–63
, pleural fluid
64
and saliva
65
. Genetic and epigenetic modifications of cfDNA
