Cancer Cell

Review

Liquid Biopsy-Based Biomarkers

of Treatment Response and Resistance
Elaine Kilgour,1,2 Dominic G. Rothwell,1,2 Ged Brady,1,2 and Caroline Dive1,2,*
1Cancer Research UK Manchester Institute Cancer Biomarker Centre, University of Manchester, Alderley Park, Macclesfield SK10 4TG, UK
2Cancer Research UK Lung Cancer Centre of Excellence, University of Manchester, Oxford Road, Manchester M13 9PL, UK
*Correspondence: caroline.dive@manchester.ac.uk
https://doi.org/10.1016/j.ccell.2020.03.012

Predictive biomarkers aid selection of personalized therapy targeted to molecular alterations within an indi-
vidual’s tumor. Patients’ responses to targeted therapies are commonly followed by treatment resistance.
Here, we survey liquid biopsies as alternatives to tumor biopsies to assess predictive and therapy response
biomarkers. We examine the potential of liquid biopsies to meet the challenges of minimal residual disease
monitoring after curative intent treatment for earlier detection of disease recurrence. We focus on blood, the
most commonly collected minimally invasive clinical sample, and on the two most widely studied assays,
circulating tumor DNA and circulating tumor cells.

Introduction
In the era of precision oncology, molecular profiling of individual
patients’ tumors can direct treatment decisions, monitor subse-
quent responses, and alert to emergence of treatment resistance
and disease relapse. Gold standard molecular profiling rests with
resected or biopsied tumors. But, is the tide gradually turning to-
ward liquid biopsies? The term liquid biopsy refers to sampling of
analytes from various biological fluids, usually blood, but
including other clinical specimens such as urine, ascites, and ce-
rebrospinal fluid (Pantel and Alix- Panabieres, 2010). Challenges
of tumor acquisition (to mention a few) are tumor accessibility,
limited specimen quantity and quality, timing of biopsy relative
to treatment selection, and difficulties in acquiring tissue biopsy
samples longitudinally to gauge disease response or monitor
relapse. Tumor heterogeneity is also problematic: how well
does a small biopsy sample represent the whole tumor? In pa-
tients with multiple metastases, how many tumor biopsy sam-
ples from different anatomic sites should be harvested and their
molecular profiles compared to gain a holistic view of the dis-
ease? Since the blood bathes most tumor sites in patients with
advanced cancers, it is perhaps not unreasonable to speculate
that blood-based liquid biopsies might better reflect tumor het-
erogeneity than small tumor biopsies and that circulating tumor
cell (CTC) analysis is likely to enrich for the most dangerous can-
cer clones that seed metastatic and often incurable disease.
The aforementioned practical and scientific issues of tumor
sampling have provoked increasing interest in the development
of minimally invasive liquid biopsies, implemented both at pre-
treatment baseline and longitudinally as predictive biomarkers,
to monitor response and anticipate treatment resistance. Circu-
lating proteins, including prostate-specific antigen, carbohy-
drate antigen 19-9 (ca19-9), and carcinoembryonic antigen
(CEA), have been in routine clinical use as tumor markers for de-
cades. However, in today’s precision oncology era, liquid bi-
opsies now frequently involve extraction and analysis of circu-
lating tumor DNA (ctDNA), a fraction of circulating free DNA
(cfDNA) in plasma to assess somatic mutations, copy number al-
terations, and gene fusions with which to select targeted thera-
pies and assess their impact (Figure 1). CTCs, cells that have ac-
quired the ability to migrate from the primary tumor, invade
through tissue, and enter the bloodstream, although more tech-
nically challenging, afford a broader scope for biomarker devel-
opment (Figure 1). Tumor exosomes, tumor educated platelets
(TEPs), circulating free RNA (cfRNA), and micro RNAs (cmiRNA)
are under evaluation as liquid biopsy specimens, although few
clinically validated tests exist for prediction or monitoring of ther-
apy responses (Heitzer et al., 2019). Most recently, the introduc-
tion of immunotherapy with durable responses in subsets of can-
cer patients heralded the search for much needed predictive
biomarkers with which to stratify patients. This in turn raised
the question of whether a tumor biopsy to study the local im-
mune landscape was mandatory for predictive biomarker dis-
covery or whether a biomarker could reasonably be sought in a
peripheral blood sample? Early data, discussed below, suggest
that the latter may be feasible via analysis of tumor mutation
burden (TMB) in ctDNA and molecular profiling of peripheral
T cells, potentially adding a new liquid biopsy candidate to a
growing list of blood tests for management of cancer patients.
Circulating Tumor DNA as a Clinical Biomarker
The presence of cfDNA in human blood was first described over
50 years ago (Mandel and Metais, 1948; Tan and Kunkel, 1966)
and defines various forms of degraded DNA fragments that are
circulating freely in the bloodstream. The majority of cfDNA is
usually derived from normal healthy leukocytes and stromal
cells, however in cancer patients, cfDNA can also include a tu-
mor-derived fraction, termed ctDNA. The use of preservative
blood tubes is an important pre-analytical consideration
because it minimizes rupture of white blood cells and hence
the dilution of ctDNA in the sample. The first clinical application
of cfDNA was pioneered by Dennis Lo with the detection of cell-
free fetal DNA (cffDNA) in non-invasive prenatal testing (NIPT) (Lo
et al., 1997; Wong and Lo, 2016); today there are millions of NIPT
tests taken worldwide with minimal risk compared with tradi-
tional sampling of amniotic fluid or placenta. The amount of
cfDNA in the blood of healthy individuals is variable, but is typi-
cally 2000 haploid genome equivalents/mL (Phallen et al.,
2017). However, cfDNA levels are often higher in cancer patients
(Leon et al., 1977; Shapiro et al., 1983), and numerous studies

Cancer Cell 37, April 13, 2020 ª 2020 Published by Elsevier Inc. 485
 Cancer Cell

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Figure 1. Clinical Biomarker Applications of Circulating Tumor DNA and Circulating Tumor Cells
Mutations and copy number alterations (CNA) can be measured in circulating tumor DNA (ctDNA) and are increasingly used to predict response to targeted
therapies and assess treatment responses. More recently, measurement of methylated ctDNA is emerging as a sensitive assay to complement mutation and CNA
analysis in ctDNA and may provide an indication of tumor site of origin for example in cancer of unknown primary. Longitudinal monitoring of ctDNA mutations is
being used to track emergent therapy resistance and identify resistance mechanisms. Studies monitoring minimal residual disease after curative intent treatment
with ctDNA to pick up disease relapse early are showing promise. Beyond circulating tumor cell (CTC) enumeration for prognosis, CTCs offer the opportunity to
measure proteins, notably of PD-L1 and ARV7 expression, to assist treatment selection. Genomic analysis of single CTCs facilitates studies of tumor hetero-
geneity and evolution. Longitudinal enumeration of CTCs can also be used to monitor treatment responses. CTCs can, in some tumor types, be harvested to
generate patient-derived explant (CDX) models in immune-deficient mice supporting drug and biomarker development. Current efforts are ongoing to optimize
direct CTC cultures that could facilitate treatment testing in real time with potential to inform clinical decision making.

report correlations between tumor burden, disease stage, and
cfDNA concentration. This was highlighted in a 640 pan-cancer
patient study with significant differences in cfDNA levels seen
between cancer types, and the median cfDNA concentration
was shown to be 100-fold higher in patients with stage IV versus
stage I disease (Bettegowda et al., 2014). The process of ctDNA
release from tumor cells has been associated with apoptotic and
necrotic cell death and via active processes in living cells (Thierry
et al., 2016; Stroun et al., 2001), yet the biology of ctDNA is not
well understood and warrants detailed investigation. Despite
its potential, ctDNA is not necessarily applicable to all cancers;
some tumor types are bad ctDNA shedders (e.g., gliomas and
sarcomas), an obvious obstacle for ctDNA profiling. The reason
for low ctDNA levels is unclear, but is thought to be associated
with tumor vascularity, location (e.g., bone lesions), or the
blood-brain barrier (Bronkhorst et al., 2019). Another significant
challenge associated with highly sensitive cfDNA analysis is
the presence of somatic mutations that accumulate in hemato-
poietic cells during aging that can then drive clonal expansion,
termed clonal hematopoietic mutations of indeterminate poten-
tial (CHIP) (Steensma et al., 2015). As the majority of cfDNA de-
rives from hematopoietic cells, these CHIP mutations are picked
up in plasma and, without appropriate controls, can be inaccu-
rately attributed to tumor. High-sensitivity cfDNA approaches
have identified CHIP mutations in 60%–90% of individuals
without cancer and shown them to be more prevalent in older in-
dividuals (Liu et al., 2019; Razavi et al., 2019). To avoid calling
these false-positive CHIP mutations, white blood cell controls,
CHIP-associated variant filtering, and deep-error controlled
sequencing are required. Despite these limitations, the ease

486 Cancer Cell 37, April 13, 2020

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and low cost of sample collection and technical advances in mu-
tation analysis has driven an increasing interest in the use of
ctDNA as a predictive biomarker (Figure 1), but are we closer
to clinical implementation?
ctDNA to Predict Response in Non-small-Cell Lung
Cancer Patients to Epithelial Growth Factor Receptor
Inhibitors
The poster child of a clinically applicable ctDNA predictive
biomarker was developed by Roche and AstraZeneca as a com-
panion diagnostic for their epithelial growth factor receptor
(EGFR) inhibitors and is described in detail in this review. About
15% of western and 40% of Asian patients with non-small-cell
lung cancer (NSCLC) harbor recurrent somatic activating muta-
tions in EGFR. These occur in exons encoding the kinase domain
and most commonly involve small in-frame deletions in exon19
(ex19del) and leucine-to-arginine point mutation at position 858
in exon 21 (L858R), resulting in EGFR activation and ligand inde-
pendency (Pao and Chmielecki, 2010). First-generation EGFR
tyrosine kinase inhibitors (TKIs) including erlotinib and gefitinib
are EGFR specific, reversible, competitive ATP inhibitors, and
an effective standard of care in previously untreated patients
harboring these mutations (Herbst et al., 2018). Although patients
typically respond well, the majority eventually relapse, usually
within 9–14 months. Profiling of these relapsed patients’ tumors
revealed that the majority acquire a second EGFR mutation, the
T790M gatekeeper, which prevents inhibition by first-generation
TKIs by increasing ATP affinity and steric hindrance (Herbst et al.,
2018; Yu et al., 2013). Osimertinib inhibits the EGFR-sensitizing
mutations and T790M and is approved for treatment of T790M
mutant patients progressed on EGFR TKI treatment and more
recently, for first-line treatment of EGFR mutant NSCLC (Herbst
et al., 2018; Ja€nne et al., 2015). The development of these tar-
geted therapies have made somatic EGFR mutation testing
essential in this patient cohort. However, obtaining tumor biopsy
samples from patients with advanced NSCLC is often chal-
lenging, especially at relapse due to poor patient performance
status and tumor location, thus heightening interest in a liquid bi-
opsy. Studies utilizing blood samples, along with matched tumor
tissue as a non-reference standard, showed high sensitivity
(72%–87%) and specificity (97%–98%) for L858R and exon19
deletion EGFR mutations using the cobas real-time PCR (RT-
PCR) platform (Roche Molecular Systems) (Thress et al., 2015;
Jenkins et al., 2017; Wu et al., 2018). Analysis of samples from
the erlotinib phase III ENSURE trial in first-line stage IIIB/IV
NSCLC patients showed that plasma was positive in 77% of tis-
sue-positive cases and negative in 98% of tissue-negative cases
(Kwapisz, 2017). In 2016, these data led to US Food and Drug
Administration (FDA) approval of the cobas EGFR Mutation
Test v2 for detection of EGFR ex19del or L858R mutations from
plasma, making this the first approved ctDNA-based companion
diagnostic test (Kwapisz, 2017). Sensitivity (61%–73%) and
specificity (67%–79%) for the T790M resistance mutation in
plasma by cobas are lower than for L858R and ex19del mutations
(Thress et al., 2015; Jenkins et al., 2017; Oxnard et al., 2016),
most likely due to T790M being an acquired mutation and so pre-
sent at lower abundance than driver activating mutations. Subse-
quent studies have shown that a positive liquid biopsy result is
more likely in patients with extra-thoracic disease with a higher
baseline tumor burden and in later lines of therapy, consistent
with greater shedding of ctDNA (Jenkins et al., 2017). However,
the clinical response rate to osimertinib in patients positive for
plasma T790M is identical to that in those positive in a tumor bi-
opsy (Thress et al., 2015; Oxnard et al., 2016). Other platforms
can also be used for ctDNA EGFR mutation detection, including
BEAMing, droplet digital (ddPCR), and next-generation
sequencing (NGS) with excellent concordance between plat-
forms (Thress et al., 2015; Jenkins et al., 2017; Oxnard et al.,
2016). The overall recommendation is that patients testing posi-
tive for EGFR mutation in plasma are eligible for an EGFR TKI,
whereas a negative plasma result should be confirmed in tumor
tissue whenever possible. These approvals, the first for ctDNA
testing, brought liquid biopsies into clinical practice, increasing
accessibility to EGFR mutation analysis in lung cancer patients
with no available tumor biopsy material, and paves the way for
further implementation of liquid biopsy-based predictive bio-
markers.
ctDNA to Monitor Treatment Response Using Targeted
Approaches
As described for EGFR mutation detection in NSCLC, in tumor
types where a limited number of recurrent driver mutations are
prevalent, such as BRAF-mutated melanomas (Schadendorf
et al., 2018) or KRAS mutant pancreatic cancers (Kamisawa
et al., 2016), ctDNA analysis across a small gene panel can serve
as a sensitive liquid biopsy. These small, targeted approaches
usually include analysis of 1–5 genes and utilize RT-PCR and
ddPCR where small numbers of variants are tested to high sensi-
tivity (variant allele frequencies [VAFs] down to 0.001% with suffi-
cient input DNA). This has application for patient monitoring after
treatment, particularly when combined with a baseline tumor bi-
opsy to confirm the presence of the dominant driver mutations
(Figure 1). The Bardelli laboratory first demonstrated that cfDNA
could be used to track these lethal clones during treatment of
colorectal cancer (CRC) with EGFR-targeted therapies (Siravegna
et al., 2015). Detection of RAS mutations in cfDNA using ddPCR
and BEAMING correlated with resistance to EGFR blockade,
and interestingly, mutated RAS clones declined upon withdrawal
of treatment, indicating that dynamic clonal evolution is driven by
treatment. This was demonstrated further by Parseghian et al.
(2019), confirming that following anti-EGFR treatment withdrawal,
RAS and EGFR mutant CRC clones lack a growth advantage rela-
tive to other clones and decay, with a cumulative half-life of
4.4 months. These data suggest that targeted cfDNA monitoring
could guide optimal timing of anti-EGFR re-challenge in CRC.
Another example of patient monitoring with ctDNA across a small
gene panel is the ongoing CACTUS trial in advanced cutaneous
melanoma (https://clinicaltrials.gov/ct2/show/NCT03808441).
This study measures circulating BRAF mutation levels with rapid
turnaround of a ddPCR ctDNA test looking at just three mutations
(V600E, V600K, and V600R) with the result used to instruct treat-
ment switching from targeted therapy to immunotherapy.
ctDNA to Monitor Treatment Response with Broad
Panels and Genome-wide Approaches
Analysis of larger gene panels (10–100s of genes) or genome-
wide approaches are required when a broader spectrum of clin-
ically important mutations are to be examined or where prior
knowledge of tumor-associated mutations is not available.
Cancer Cell 37, April 13, 2020 487

Analysis of such comprehensive panels in ctDNA ddPCR and
RT-PCR is not feasible, and NGS approaches are required.
The power of combining cfDNA analysis with NGS to detect so-
matic mutations in cancer patients across multiple genes was
first described in 2012 by Forshew et al. (2012). The approach,
termed TAm-Seq, enabled analysis of nearly 6,000 genomic ba-
ses at VAFs as low as 2%, greatly increasing the scope of ctDNA
detection (Forshew et al., 2012). A similar approach to detect
tumor-specific chromosomal alterations was described by the
Velculescu laboratory in the same year (Leary et al., 2012). The
clinical utility of cfDNA NGS approaches has also been demon-
strated in serial plasma samples to track genomic evolution of
metastatic cancers in response to therapy in advanced breast,
ovarian, and lung cancers (Murtaza et al., 2013; Leary et al.,
2012). To facilitate the use of ctDNA as a biomarker across
more tumor types and disease stages, the issues of false-posi-
tive (mutations being incorrectly called due to technical error
and noise) and false-negative (mutations being missed due to
a lack of sensitivity of the assay) variant calling as well as
CHIP, as described earlier, needs to be overcome. This has
involved considerable efforts to increase the sensitivity of cfDNA
NGS, including the addition of unique molecular identifiers (UMI)
and digital error suppression (iDES) (Newman et al., 2014, 2016).
UMIs enable every cfDNA fragment to be uniquely identified and
a true consensus sequence of each molecule determined,
thereby reducing false-positive variant calling. The combination
of UMIs and iDES in the CAPPseq workflow correctly identified
EGFR mutations in 66 NSCLC samples with 92% sensitivity
and 96% specificity, with detection of ctDNA down to 4 in 105
cfDNA molecules. A similar approach, TEC-Seq (targeted error
correction sequencing) also incorporating UMIs detected ctDNA
across a panel of 58 cancer-associated genes in 200 early-stage
cancer patients (Phallen et al., 2017) with mutations detected in
59%–71% of stage I or II patients, depending on disease type. In
addition to the development of UMIs and improved bioinformatic
approaches, increased sensitivity of ctDNA detection has also
been described based on observations that ctDNA fragments
differ in length from cfDNA from healthy cells; typically 90–
150 bp or >250 bp compared with 166 bp, respectively. These
differences can be exploited both technically and bio-
informatically (Underhill et al., 2016; Mouliere et al., 2018;
Cristiano et al., 2019). Using an in vitro and in silico approach
to select fragments between 90 and 150 bp, Mouliere et al.
(2018) demonstrated a >2-fold median enrichment in detection
of tumor DNA in >95% of 200 pan-cancer patients, where
analysis of size-selected cfDNA identified somatic mutations in
samples that were otherwise undetectable. Physical selection
of low-frequency ctDNA is technically challenging, however in
silico analysis of differences in fragmentation patterns from
shallow whole-genome sequencing (sWGS) also increased the
sensitivity of ctDNA detection in 236 patients encompassing
seven cancer types with sensitivities from 57% to >99% and a
specificity of 98% (Cristiano et al., 2019).
In addition to improved mutational analysis, methylation
profiling of cfDNA also has potential to increase ctDNA detection
sensitivity by encompassing tissue- and cancer-specific large-
scale epigenetic alterations (Figure 1). As cancer methylome
mapping expands, DNA methylation profiles can differentiate tu-
mor and normal tissues. Four common cancers were differenti-
488 Cancer Cell 37, April 13, 2020
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ated from normal tissue with >95% accuracy, and their methyl-
ation profiles correctly identified 19 of 20 breast cancer
metastases from normal breast and 29 of 30 CRC metastases
from liver biopsies (Hao et al., 2017). However, conventional
methylation profiling typically requires micrograms of DNA and
bisulfite conversion, which destroy non-methylated DNA (Bhat-
tacharjee et al., 2018), and so is not compatible with the limited
amounts of material typically associated with a liquid biopsy. The
potential of methylation profiling of cfDNA was recently demon-
strated using a novel immunoprecipitation-based protocol that
detected target cfDNA down to 0.001% (compared with 0.1%
for a previously used hybridization detection approach) (Shen
et al., 2018). A similar approaching analyzing transcription start
sites where nucleosome occupancy results in different read
depth coverage patterns for expressed and silent genes has
also been described (Ulz et al., 2016). This approach found anal-
ysis of cfDNA from metastatic patients could classify expressed
cancer driver genes in regions with somatic copy number gains
with high accuracy. This has been further refined to infer acces-
sibility of transcription factor binding sites from cell-free DNA
fragmentation patterns that enables accurate prediction of tumor
subtypes in prostate cancer (Ulz et al., 2019) with future potential
for predictive biomarker development.
NGS ctDNA Assays with Broadened Scope to Select
Targeted Therapies
With the development of these high-sensitivity, high-specificity
ctDNA assays, a broadened scope of ctDNA as a predictive
biomarker with clinical utility is being realized (Figure 1).
Large-scale retrospective validation studies evaluating concor-
dance with tumor mutations show promising results. As an
exemplar, a recent study of 10,593 patients showed >99.6%
technical success and 85.9% sensitivity using the Guardant360
cfDNA assay, which covers 73 cancer-related genes (Ode-
gaard et al., 2018). This study also reported concordance be-
tween cfDNA assay and tissue-based clinical genotyping
across >750 patients with positive percentage agreement and
negative percentage agreement levels >99%. To move ctDNA
profiling into the clinical mainstream, prospective studies to
validate the clinical utility of NGS ctDNA assays are required.
One such study is the TARGET study, (Rothwell et al., 2019)
which aims to match patients with a broad range of advanced
cancers to early-phase clinical trials based on analysis of a
641 cancer-associated-gene cfDNA assay. Analysis of the first
100 patients showed that somatic mutation calling from cfDNA
could be performed, and data reported to a molecular tumor
board in a clinically feasible timescale showed good concor-
dance with tissue (78% concordance) and identified potentially
actionable mutations in 41% of patients (Rothwell et al., 2019).
Of these patients 11 of 41 (27%) went on to a match therapy,
which reflects a similar level of recruitment to conventional tis-
sue-based targeted approaches and does not reflect a failing of
ctDNA analysis, rather the insufficiency of targeted therapy
drugs for detected and potentially targetable mutations (Tan-
nock and Hickman, 2019).
Minimal Residual Disease Monitoring and Anticipation
of Disease Recurrence Using ctDNA
A more technically challenging approach using ctDNA is its
deployment as an early warning of relapsing disease after
Cancer Cell
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treatment with curative intent, which could direct adjuvant ther-
apy decision making or earlier intervention (Figure 1). The hy-
pothesis is that ctDNA reflects tumor burden, and its level in-
creases as tumor regrowth occurs. The obvious advantage of
the approach is the ease of longitudinal blood sample collection.
One of the first studies to highlight the utility of ctDNA in minimal
residual disease (MRD) monitoring was reported by Nick Turn-
er’s group following analysis of a prospective cohort of 55
early-stage breast cancer patients at high risk of relapse (Gar-
cia-Murillas et al., 2015). ctDNA detection at completion of treat-
ment predicted metastatic relapse with high accuracy, and mu-
tation tracking in serial samples detected disease relapse
7.9 months earlier than clinical surveillance (Garcia-Murillas
et al., 2015). More recently, the same group has shown that, in
a cohort of early-stage breast cancer patients, detection of
ctDNA during follow-up was associated with relapse (hazard ra-
tio [HR], 25.2) and detection of ctDNA at diagnosis was also
associated with relapse-free survival (HR, 5.8). In this cohort,
ctDNA detection had a median lead time of 10.7 months (Gar-
cia-Murillas et al., 2019). Similar studies showing an association
between disease-free survival and detection of ctDNA post
definitive treatment in numerous tumor types, including CRC,
breast carcinoma, pancreatic carcinoma, and lung carcinoma,
have also been reported (reviewed in Coakley et al., 2019). As
discussed earlier, mutation panels can be tailored to each pa-
tient informed by sequencing their tumor with selection of driver,
truncal mutations for highly sensitive MRD monitoring. Tracking
multiple mutations rather than a single point mutation also in-
creases the sensitivity of ctDNA detection for MRD (Newman
et al., 2014), an approach deployed in the TRACERx study of
NSCLC evolution (Abbosh et al., 2017). In this study, which fol-
lows NSCLC patients after curative intent surgery, generation
of patient-specific multiplexed PCR panels targeting a median
of 18.5 or 28 mutations per patient in lung adenocarcinoma
and squamous cell carcinoma patients, respectively, detected
at least two SNVs in 13 of 14 (93%) patients prior to, or at, clinical
relapse with a median interval between ctDNA detection and
NSCLC relapse by computed tomography imaging of 70 days
(Abbosh et al., 2017). This approach has been further exempli-
fied by the multiplexed targeted digital sequencing (TARDIS)
approach, which analyzed up to 115 mutations per patient
from 33 women with stage I to III breast cancer, resulting in sen-
sitivities of 91% and 53% at VAFs of 3 in 104 and 3 in 105,
respectively (McDonald et al., 2019). The sensitivities of these
techniques and promising initial results suggest that personal-
ized ctDNA tracking has great potential in the clinical manage-
ment of patients treated with curative intent.
In summary, there is mounting support for the use of ctDNA as
a clinically applicable predictive biomarker of treatment
response and resistance in cancer (Figure 1). There are
numerous approaches available, including targeted small-panel
digital PCR, which enables quantitative measurement of limited
numbers of mutations at sensitivities down to 0.001%, for a rela-
tively low cost. The other extreme includes NGS-based broad-
panel and whole-genome approaches such as methylation
profiling, which tend to be semi-quantitative, with poorer sensi-
tivities and higher costs, but which have the potential to provide
much more information. The appropriate approach will depend
on the clinical question being asked.
Circulating Tumor Cells as Clinical Biomarkers and to
Support Drug Development
Although technical challenges remain for ctDNA assay develop-
ment, it has become the ‘‘go to’’ liquid biopsy in clinical trials,
with an avalanche of studies reporting encouraging data on its
clinical utility as a predictive and response biomarker and an
MRD monitoring tool. In contrast, CTCs have only recently
gained substantial traction beyond their FDA-approved use a
decade ago as prognostic biomarkers in breast cancer, prostate
cancer, and CRC (Cristofanilli et al., 2004; Cohen et al., 2008; De
Bono et al., 2008, Figure 1). The primary obstacle to further clin-
ical implementation has been the high technical bar of rare and
phenotypically heterogeneous tumor cell detection, enrichment,
and isolation with typically only 1 CTC/mL of blood (Miller et al.,
2010). Nevertheless, as CTC platforms evolve and improve (Pan-
tel and Alix- Panabieres, 2019), attaining the full potential of CTC
analyses, capturing DNA, RNA, and protein readouts, remains
both tantalizing and pertinent as evidence mounts for therapy
resistance mechanisms not readily measureable in ctDNA, for
example, via phenotype switching. Moreover, while ctDNA con-
centration is commonly associated with tumor burden (a balance
of tumor cell proliferation and death), mechanisms governing tu-
mor cell invasion, migration, and extra- and intravasation do not
evoke an obligatory link to tumor size; small primary tumors can
metastasize, and large tumors do not always do so. The differing,
albeit incompletely understood, underlying mechanisms result-
ing in ctDNA shedding and dissemination of CTCs emphasize
the complementarity of these distinct liquid biopsy approaches.
The robust and semi-automated CellSearch platform enriches
CTCs based on EpCAM expression and identifies these cells
based on the absence of CD45 and the presence of cytokeratin
(CK) expression. CellSearch is considered the gold standard for
CTC enrichment and enumeration and is FDA approved as a
prognostic biomarker for breast cancer, prostate cancer, and
CRC (with a threshold of 5, 5, and 3 CTCs/7.5 mL of blood,
respectively) (Cristofanilli et al., 2004; Cohen et al., 2008; De
Bono et al., 2008). CellSearch CTCs are also prognostic for pro-
gression-free survival and overall survival in small-cell lung can-
cer (SCLC) and NSCLC (50 and 5 CTCs/7.5 mL of blood, respec-
tively) and can be used to monitor therapy response and relapse
in, for example, lung, breast, and prostate cancers (Hou et al.,
2009; Krebs et al., 2011; Bidard et al., 2014; Lorente et al.,
2016; Vogelzang et al., 2017; Heller et al., 2018; Pantel et al.,
2019). However, if epithelial-to-mesenchymal transition results
in downregulated EpCAM and/or CK expression, CTCs become
invisible to the CellSearch platform, and marker-independent
platforms that capture epithelial and mesenchymal CTCs will
have better sensitivity as a result (Krebs et al., 2014). Notably,
although prognostic, CellSearch CTCs were detectable in the
peripheral blood of only 32% of 101 metastatic NSCLC patients
(Krebs et al., 2011). Several platforms for marker-independent
CTC enrichment based on size and physical properties are under
evaluation (Krebs et al., 2014; Chudziak et al., 2016; Janning
et al., 2019) but are not yet approved as clinical tests. CTC isola-
tion technologies (such as DEPArray@ or ALS@) can be imple-
mented after CTC enrichment. However, any enrichment step
is likely to result in lost CTCs. The ‘‘no cell left behind’’ platform
from EPIC Sciences dispenses with the enrichment step and
captures all nucleated cells in a blood sample onto slides,
Cancer Cell 37, April 13, 2020 489

supporting analysis of a broader range of CTC types as exempli-
fied in prostate cancer and NSCLC (Werner et al., 2015; McDa-
niel et al., 2017; Boffa et al., 2017). Captured cells are interro-
gated with multiplexed immunofluorescence assays (including
exclusion and tumor markers) with proprietary algorithms to pre-
sent candidate tumor cells (Keomanee-Dizon et al., 2020), and
physical picking of such cells for downstream genomic analysis
can confirm tumor origin. While ctDNA is becoming a routine
approach to assess tumor mutations and copy number changes,
CTCs also offer analysis of protein expression (Figure 1) and sub-
cellular localization along with single-cell analysis of tumor het-
erogeneity (e.g., Carter et al., 2017; Chemi et al., 2019).
Predicting Therapy Responses with CTCs
The leading exemplar of an FDA-approved CTC predictive
biomarker is the detection of the constitutively active ARV7
androgen receptor splice variant associated with prostate can-
cer resistance to anti-hormonal drugs abiraterone and enzaluta-
mide using the EPIC Sciences platform (Figure 1). Nuclear ARV7
expression in patient CTCs predicts better outcome on taxane
and worse outcomes on anti-hormonals, validating clinical utility
of this assay (Scher et al., 2018; Pantel et al., 2019). An explor-
atory biomarker example of CTC analysis in clinical trials
involved estrogen receptor expression in breast cancer CTCs
using CellSearch alongside analysis of mutational status, which
suggested existence of multiple mechanisms of resistance to
endocrine therapy, including incomplete estrogen receptor
downregulation and emergence of estrogen receptor mutations
(Paoletti et al., 2018a, 2018b). In addition, data from breast can-
cer studies suggests that characterization of both CTCs and
ctDNA may provide a better early prediction of outcome (Paoletti
et al., 2018b). CTC assays for the immune checkpoint (IC) ligand
PD-L1 have also been developed using the EPIC Sciences, Cell
Search, and other platforms to explore their potential as predic-
tive biomarkers for IC targeted therapies (Mazel et al., 2015;
Boffa et al., 2017; Guibert et al., 2018; Yue et al., 2018; Janning
et al., 2019, Figure 1).
As a proof-of-principle example of genomic analysis of CTCs,
a copy number alteration (CNA) signature predictive at baseline
of subsequent chemosensitivity was reported for SCLC; this
CTC CNA classifier correctly assigned 83% of 31 cases as che-
mosensitive or chemorefractory (Carter et al., 2017). Clearly, a
larger study is required to validate the CTC classifier, and its clin-
ical implementation will be warranted only when improved ther-
apies beyond platinum-based chemotherapy are developed that
improve outcomes for chemorefractory SCLC patients. Howev-
er, this approach illustrates the potential for molecular character-
ization of CTCs to provide predictive biomarkers that inform
treatment decisions as well as mechanistic insight into drug
sensitivity and resistance.
Several groups have demonstrated that CTCs can be cultured,
notably from breast and prostate cancer patients (e.g., Zhang
et al., 2013; Yu et al., 2014; Kolostova et al., 2014), with potential
for real-time drug testing to predict donor patient responses with
robust assays tailored to low cell numbers (Figure 1). CTC cell
lines can also be derived, notably from patients with breast, colo-
rectal, and gastric cancers, and with SCLC (Yu et al., 2014; Rath
et al., 2019, Brungs et al., 2020; Soler et al., 2018; Klameth et al.,
2017) adding new tools for drug development and to explore the
biology of advanced cancers, with the caveat, as with all cell cul-
490 Cancer Cell 37, April 13, 2020
Cancer Cell
Review
tures, that long-term passage on plastic is likely to result in irre-
versible adaptation and clonal outgrowth. CTCs have also been
used to generate patient CTC Derived eXplant (CDX) models in
mice, first shown for CTCs in breast cancer patients (Baccelli
et al., 2013) and subsequently in SCLC where CDX models
reproduce the donor patient’s response to standard-of-care
chemotherapy (Hodgkinson et al., 2014; Drapkin et al., 2018,
Figure 1). Longitudinal blood samples from SCLC patients
have been used to generate paired models representing the dis-
ease before and after development of resistance to therapy, al-
lowing resistance to be studied in a scenario where post-treat-
ment tumor biopsies are rarely obtained (Drapkin et al., 2018;
Simpson et al., 2020). Most recently, single-cell RNA sequencing
of SCLC CTCs and CDX cells demonstrated the diversity of
changes that accompany acquired chemoresistance (Allison
Stewart et al., 2020). Disaggregated CDX cells can be cultured
ex vivo for short periods of time, limiting drift while allowing func-
tion-testing experimentation (Lallo et al., 2019). SCLC CDX
models are thus attractive to study treatment efficacy and resis-
tance mechanisms to support drug and biomarker development
and predict treatment responses in a disease where both CTCs
(Hou et al., 2012) and ctDNA (Mohan et al., 2020) are prevalent
facilitating liquid biopsies in clinical trials. However, the time
required to generate and passage CDX cells in mice is currently
not compatible with informing clinical decisions for individual
SCLC patients, and the immune-compromised mouse back-
ground renders them unsuitable for immunotherapy testing.
SCLC is a highly aggressive, early metastatic cancer where suc-
cessful generation of CDX models correlates with high CTC
numbers; CDX models are possible in NSCLC (e.g., Morrow
et al., 2016) and in prostate cancer (Tayoun et al., 2019), but
have not yet been reported in multiple tumor types.
Predicting Risk of Disease Recurrence with CTCs
The presence of CTCs after treatment with curative intent might
reflect the presence of micro-metastasis and portend a high risk
of disease relapse. Several breast cancer studies provide evi-
dence for this. A meta-analysis of more than 1,500 patients
with non-metastatic breast cancer reported that CTCs were
detectable in 25% of patients prior to neoadjuvant therapy and
associated with worse overall survival, disease-free survival,
and recurrence-free survival but notably not with pathological
complete response (Bidard et al., 2018). In the phase III SUC-
CESS trial in high risk non-metastatic breast cancer patients,
detection of CTCs 2 years post adjuvant chemotherapy was
associated with worse outcome (Trapp et al., 2019), and in local-
ized breast cancer, the presence of CTCs at 5 years after diag-
nosis occurred in 26 of 547 (4.8%) of patients and was associ-
ated with a higher risk of late recurrence among patients with
hormone receptor-positive breast cancer (Sparano et al., 2018).
The hypothesis that CTCs are associated with a high risk of
relapse was further tested in stage I–IIIa patients with NSCLC un-
dergoing surgery with curative intent. CellSearch CTCs were un-
detectable in the peripheral blood samples in most patients, but
EpCAM- and CK-positive cells were present in the pulmonary
vein (PV) draining the cancerous lung (Crosbie et al., 2016;
Chemi et al., 2019) immediately prior to tumor resection in a sub-
set of patients. Greater CellSearch CTC abundance in PV versus
peripheral blood is likely to be attributable to proximity to the pri-
mary tumor and the fact that PV blood is drawn upstream of
Cancer Cell
Review
capillary bed filtering. These PV circulating epithelial cells pre-
dicted the risk of lung-specific disease relapse in 100 patients
enrolled on the TRACERx study (Chemi et al., 2019). A larger pro-
spective study is required to validate this finding, but enumera-
tion of these epithelial cells in the PV at surgery may inform on
which patients should receive more frequent surveillance with
ctDNA and imaging after surgery. Of note, in a case study
when CellSearch PV CTCs were isolated and sequenced, these
early disseminating CTCs had a higher mutational overlap with
metastasis occurring 10 months later than did the resected pri-
mary tumor, suggesting early dissemination of PV CTCs is
responsible for disease relapse (Chemi et al., 2019). This illus-
trates the potential for CTCs to provide a better representation
of tumor heterogeneity than a tumor biopsy sample. It also sug-
gests that CTCs are enriched for the most aggressive cancer
cells more likely to form metastasis and hence very relevant for
therapeutic intervention in advanced stage disease. This hypoth-
esis is further supported by molecular profiling of SCLC patient
CTCs revealing increased heterogeneity on the development of
resistance to therapy (Allison Stewart et al., 2020).
Liquid Biopsy to Guide Immunotherapy
Immunotherapy has transformed cancer therapy in several tu-
mor types and patient subsets, with impressive durable re-
sponses most notably in melanoma, renal cancer, and NSCLC
(Kruger et al., 2019). Several candidate tumor-based predictive
biomarkers have emerged for IC therapy. Microsatellite insta-
bility (MSI) results in a high number of gene mutations within tu-
mors and is the first pan-cancer indication approved for IC
blockade (Le et al., 2017). Across several studies, TMB has
shown predictive value for IC inhibition, and the utility of a
blood-based TMB measurement using ctDNA is under evalua-
tion (Gandara et al., 2018; Wang et al., 2019; Peters et al.,
2019; Snyder et al., 2019). A retrospective analysis of the POP-
LAR and OAK clinical studies of the IC inhibitor atezolizumab
versus chemotherapy in NSCLC showed an association of
ctDNA TMB with clinical outcome, with a TMB >16 mut/Mb
demonstrating a median overall survival of 13.5 months for ate-
zolizumab versus 6.8 months with docetaxel in the OAK study
(Gandara et al., 2018). Although the MYSTIC study of IC inhibi-
tors durvalumab plus tremelimumab in first-line NSCLC failed
to meet its primary endpoint, improved benefit for the immuno-
therapy combination versus chemotherapy was observed at
higher blood-based TMB cut-offs (Peters et al., 2019). Assays
for measurement of MSI in ctDNA have also been reported
(Georgiadis et al., 2019; Willis et al., 2019), raising the opportu-
nity for analysis of both TMB and MSI from a blood sample to
predict clinical benefit from IC therapy. Studies also indicate
that changes in ctDNA can provide an early biomarker of
response to IC therapy and in long-term responders, monitoring
of ctDNA can detect MRD and predict relapse (Hellmann et al.,
2020; Goldberg et al., 2018; Anagnostou et al., 2019; Moding
et al., 2020).
Recently, it was shown that expansion of T cell clones within
the tumor is paralleled by their expansion within the peripheral
blood, and patients with such T cell clonotypic expansion
respond better to PD1 checkpoint blockade (Wu et al., 2020).
This new finding represents an important observation, high-
lighting the potential for detection of responding patients
through analysis of T cell subsets in a peripheral blood sample,
using approaches such as T cell receptor sequencing. Consis-
tent with this are recent reports on blood-based early response
biomarkers for IC blockade in melanoma patients. Valpione
et al. (2020) reported increases in productive T cell receptor se-
quences in cfDNA and the expansion of a subset of cytotoxic
memory effector T cells. Fairfax et al. (2020) reported that a
higher number of large T cell clones are detected in peripheral
blood from responding patients. Together, analysis of ctDNA
and T cell expansion represent promising developments in the
hunt for liquid biopsies to inform the use of immunotherapies.
CTC enumeration has not yet been explored as a predictive
and/or response marker for IC therapy, but assays are available
for analysis of PD-L1 expression in CTCs (Mazel et al., 2015;
Boffa et al., 2017; Guibert et al., 2018; Yue et al., 2018; Janning
et al., 2019) and their prognostic and predictive value is under
investigation.
Conclusion
The field of liquid biopsies is rapidly evolving. Most notably,
ctDNA-based biomarkers to predict and monitor tumor re-
sponses to treatment and to monitor MRD have generated
enormous interest; research publications continue to demon-
strate the feasibility of ctDNA testing in a broad range of tumor
types (Figure 1). Numerous diagnostic companies now offer
liquid biopsy testing, and drug developers are increasingly
developing ctDNA companion diagnostics for their targeted
therapies. As a result, many oncologists are embracing the
ease of blood sample collection and rapid turnaround time of
ctDNA test results, adding this approach to assist in their treat-
ment decision making. Tumor biopsies, for the most part,
remain the gold standard, but as the sensitivity and specificity
of liquid biopsy assays continue to improve and real-world ev-
idence continues to mount, it would seem that ctDNA-based
liquid biopsies are heading toward mainstream in precision
oncology. The technical challenges of CTCs remain, and
development of robust CTC assays that capture the required
phenotypic diversity takes a considerable time. However, in in-
stances where the genome alone cannot predict treatment
response and downstream information, e.g., on drug-target
hitting, is informative, a robust CTC assay, such as the AR
variant in prostate cancer, comes to the fore (Figure 1). Given
the relative ease of ctDNA assay development, CTC assay de-
velopers should perhaps double down on what ctDNA cannot
achieve in the biomarker space. The common problem of tar-
geted therapy resistance makes continuous monitoring of a
patient’s treatment response mission critical, and here liquid
biopsies will play a significant role, anticipating when to switch
treatment (Figure 1). The liquid biopsy sensitivity challenges of
MRD monitoring are beginning to be met, and a combined pre-
treatment tumor biopsy with subsequent serial liquid biopsies
based on each patient’s tumor profile form the basis of trials
to test whether patient benefit follows earlier intervention
based on ctDNA warning of relapse after curative intent treat-
ment. The biomarker promise of TEPs, tumor exosomes, and
circulating RNAs have yet to be realized in the clinic, but
ongoing translational research raises expectations that new
liquid biopsies are on the horizon, adding to the battery of
blood tests to support future precision oncology.
Cancer Cell 37, April 13, 2020 491

The next frontier for liquid biopsies is perhaps to answer the
question of whether or not they might assist with the pressing
need for predictive biomarkers for immunotherapy—or is that a
step too far, requiring an in-depth understanding of the tumor mi-
cro-environment and thus a mandatory tumor biopsy? TMB in
ctDNA, IC protein in CTCs (Figure 1), receptor sequencing of pe-
ripheral T cell subsets are all in play; their clinical utility remains to
be defined, but the recent developments suggesting liquid bi-
opsies could be developed to support immunotherapy decision
making are an exciting ‘‘watch this space.’’ The application of
liquid biopsies for early detection of cancers is a topic of huge in-
terest and importance; while many of the issues discussed here
that touch upon sensitivity and specificity of liquid biopsies are
particularly germane, early detection is beyond the remit of this
review.
The universal replacement of tumor biopsies with liquid bi-
opsies seems unrealistic; however, as ctDNA, CTC, and the
additional aforementioned blood tests improve, it seems likely
that they will become an increasingly used tool for cancer patient
management, especially when serial assessment of patients is
required and/or an invasive tumor biopsy is not feasible.
ACKNOWLEDGMENTS
E.K., D.R., G.B., and C.D. are funded by Core Funding to CRUK Manchester
Institute, United Kingdom (A27412); and the Manchester CRUK Centre Award,
United Kingdom (A25254). Support was received from the CRUK Manchester
Experimental Cancer Medicines Centre, United Kingdom (A25146); the CRUK
Lung Cancer Centre of Excellence, United Kingdom (A29420); and the Man-
chester NIHR Biomedical Research Centre, United Kingdom. We thank Ekram
Aidaros-Talbot for assistance with this manuscript. Declaration of Interests
Research funding has been received by C.D. from AstraZeneca, United
Kingdom; Astex Pharmaceuticals, United States; Bioven, United Kindom;
Amgen, United States; Carrick Therapeutics, Republic of Ireland; Merck AG,
Germany; Taiho Oncology, Japan; Biolidics Limited, Singapore; Angle PLC,
United Kingdom; Menarini Diagnostics, Italy; GSK, United Kingdom; Bayer,
Germany; Boehringer Ingelheim, Germany; Roche, Switzerland; BMS, United
States; Novartis, Switzerland; Celgene, United States; Thermo Fisher, United
States. C.D. is on the advisory board and has received consultancy fees/
honoraria from AstraZeneca, United Kingdom and Biocartis, Belgium.
DECLARATION OF INTERESTS
Research funding has been received by C.D. from AstraZeneca, Astex Phar-
maceuticals, Bioven, Amgen, Carrick Therapeutics, Merck AG, Taiho
Oncology, Clearbridge Biomedics, Angle PLC, Menarini Diagnostics, GSK,
Bayer, Boehringer Ingelheim, Roche, BMS, Novartis, Celgene, Thermo Fisher.
C.D. is on the advisory board and has received consultancy fees/honoraria
from AstraZeneca and Biocartis.
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