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

Cancer Cell 37, April 13, 2020 ª 2020 Published by Elsevier Inc. 485
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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 blood-brain barrier (Bronkhorst et al., 2019). Another significant
cfDNA concentration. This was highlighted in a 640 pan-cancer challenge associated with highly sensitive cfDNA analysis is
patient study with significant differences in cfDNA levels seen the presence of somatic mutations that accumulate in hemato-
between cancer types, and the median cfDNA concentration poietic cells during aging that can then drive clonal expansion,
was shown to be 100-fold higher in patients with stage IV versus termed clonal hematopoietic mutations of indeterminate poten-
stage I disease (Bettegowda et al., 2014). The process of ctDNA tial (CHIP) (Steensma et al., 2015). As the majority of cfDNA de-
release from tumor cells has been associated with apoptotic and rives from hematopoietic cells, these CHIP mutations are picked
necrotic cell death and via active processes in living cells (Thierry up in plasma and, without appropriate controls, can be inaccu-
et al., 2016; Stroun et al., 2001), yet the biology of ctDNA is not rately attributed to tumor. High-sensitivity cfDNA approaches
well understood and warrants detailed investigation. Despite have identified CHIP mutations in 60%–90% of individuals
its potential, ctDNA is not necessarily applicable to all cancers; without cancer and shown them to be more prevalent in older in-
some tumor types are bad ctDNA shedders (e.g., gliomas and dividuals (Liu et al., 2019; Razavi et al., 2019). To avoid calling
sarcomas), an obvious obstacle for ctDNA profiling. The reason these false-positive CHIP mutations, white blood cell controls,
for low ctDNA levels is unclear, but is thought to be associated CHIP-associated variant filtering, and deep-error controlled
with tumor vascularity, location (e.g., bone lesions), or the sequencing are required. Despite these limitations, the ease

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and low cost of sample collection and technical advances in mu- with greater shedding of ctDNA (Jenkins et al., 2017). However,
tation analysis has driven an increasing interest in the use of the clinical response rate to osimertinib in patients positive for
ctDNA as a predictive biomarker (Figure 1), but are we closer plasma T790M is identical to that in those positive in a tumor bi-
to clinical implementation? opsy (Thress et al., 2015; Oxnard et al., 2016). Other platforms
ctDNA to Predict Response in Non-small-Cell Lung can also be used for ctDNA EGFR mutation detection, including
Cancer Patients to Epithelial Growth Factor Receptor BEAMing, droplet digital (ddPCR), and next-generation
Inhibitors sequencing (NGS) with excellent concordance between plat-
The poster child of a clinically applicable ctDNA predictive forms (Thress et al., 2015; Jenkins et al., 2017; Oxnard et al.,
biomarker was developed by Roche and AstraZeneca as a com- 2016). The overall recommendation is that patients testing posi-
panion diagnostic for their epithelial growth factor receptor tive for EGFR mutation in plasma are eligible for an EGFR TKI,
(EGFR) inhibitors and is described in detail in this review. About whereas a negative plasma result should be confirmed in tumor
15% of western and 40% of Asian patients with non-small-cell tissue whenever possible. These approvals, the first for ctDNA
lung cancer (NSCLC) harbor recurrent somatic activating muta- testing, brought liquid biopsies into clinical practice, increasing
tions in EGFR. These occur in exons encoding the kinase domain accessibility to EGFR mutation analysis in lung cancer patients
and most commonly involve small in-frame deletions in exon19 with no available tumor biopsy material, and paves the way for
(ex19del) and leucine-to-arginine point mutation at position 858 further implementation of liquid biopsy-based predictive bio-
in exon 21 (L858R), resulting in EGFR activation and ligand inde- markers.
pendency (Pao and Chmielecki, 2010). First-generation EGFR
tyrosine kinase inhibitors (TKIs) including erlotinib and gefitinib ctDNA to Monitor Treatment Response Using Targeted
are EGFR specific, reversible, competitive ATP inhibitors, and Approaches
an effective standard of care in previously untreated patients As described for EGFR mutation detection in NSCLC, in tumor
harboring these mutations (Herbst et al., 2018). Although patients types where a limited number of recurrent driver mutations are
typically respond well, the majority eventually relapse, usually prevalent, such as BRAF-mutated melanomas (Schadendorf
within 9–14 months. Profiling of these relapsed patients’ tumors et al., 2018) or KRAS mutant pancreatic cancers (Kamisawa
revealed that the majority acquire a second EGFR mutation, the et al., 2016), ctDNA analysis across a small gene panel can serve
T790M gatekeeper, which prevents inhibition by first-generation as a sensitive liquid biopsy. These small, targeted approaches
TKIs by increasing ATP affinity and steric hindrance (Herbst et al., usually include analysis of 1–5 genes and utilize RT-PCR and
2018; Yu et al., 2013). Osimertinib inhibits the EGFR-sensitizing ddPCR where small numbers of variants are tested to high sensi-
mutations and T790M and is approved for treatment of T790M tivity (variant allele frequencies [VAFs] down to 0.001% with suffi-
mutant patients progressed on EGFR TKI treatment and more cient input DNA). This has application for patient monitoring after
recently, for first-line treatment of EGFR mutant NSCLC (Herbst treatment, particularly when combined with a baseline tumor bi-
et al., 2018; Ja€nne et al., 2015). The development of these tar- opsy to confirm the presence of the dominant driver mutations
geted therapies have made somatic EGFR mutation testing (Figure 1). The Bardelli laboratory first demonstrated that cfDNA
essential in this patient cohort. However, obtaining tumor biopsy could be used to track these lethal clones during treatment of
samples from patients with advanced NSCLC is often chal- colorectal cancer (CRC) with EGFR-targeted therapies (Siravegna
lenging, especially at relapse due to poor patient performance et al., 2015). Detection of RAS mutations in cfDNA using ddPCR
status and tumor location, thus heightening interest in a liquid bi- and BEAMING correlated with resistance to EGFR blockade,
opsy. Studies utilizing blood samples, along with matched tumor and interestingly, mutated RAS clones declined upon withdrawal
tissue as a non-reference standard, showed high sensitivity of treatment, indicating that dynamic clonal evolution is driven by
(72%–87%) and specificity (97%–98%) for L858R and exon19 treatment. This was demonstrated further by Parseghian et al.
deletion EGFR mutations using the cobas real-time PCR (RT- (2019), confirming that following anti-EGFR treatment withdrawal,
PCR) platform (Roche Molecular Systems) (Thress et al., 2015; RAS and EGFR mutant CRC clones lack a growth advantage rela-
Jenkins et al., 2017; Wu et al., 2018). Analysis of samples from tive to other clones and decay, with a cumulative half-life of
the erlotinib phase III ENSURE trial in first-line stage IIIB/IV 4.4 months. These data suggest that targeted cfDNA monitoring
NSCLC patients showed that plasma was positive in 77% of tis- could guide optimal timing of anti-EGFR re-challenge in CRC.
sue-positive cases and negative in 98% of tissue-negative cases Another example of patient monitoring with ctDNA across a small
(Kwapisz, 2017). In 2016, these data led to US Food and Drug gene panel is the ongoing CACTUS trial in advanced cutaneous
Administration (FDA) approval of the cobas EGFR Mutation melanoma (https://clinicaltrials.gov/ct2/show/NCT03808441).
Test v2 for detection of EGFR ex19del or L858R mutations from This study measures circulating BRAF mutation levels with rapid
plasma, making this the first approved ctDNA-based companion turnaround of a ddPCR ctDNA test looking at just three mutations
diagnostic test (Kwapisz, 2017). Sensitivity (61%–73%) and (V600E, V600K, and V600R) with the result used to instruct treat-
specificity (67%–79%) for the T790M resistance mutation in ment switching from targeted therapy to immunotherapy.
plasma by cobas are lower than for L858R and ex19del mutations
(Thress et al., 2015; Jenkins et al., 2017; Oxnard et al., 2016), ctDNA to Monitor Treatment Response with Broad
most likely due to T790M being an acquired mutation and so pre- Panels and Genome-wide Approaches
sent at lower abundance than driver activating mutations. Subse- Analysis of larger gene panels (10–100s of genes) or genome-
quent studies have shown that a positive liquid biopsy result is wide approaches are required when a broader spectrum of clin-
more likely in patients with extra-thoracic disease with a higher ically important mutations are to be examined or where prior
baseline tumor burden and in later lines of therapy, consistent knowledge of tumor-associated mutations is not available.

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Analysis of such comprehensive panels in ctDNA ddPCR and ated from normal tissue with >95% accuracy, and their methyl-
RT-PCR is not feasible, and NGS approaches are required. ation profiles correctly identified 19 of 20 breast cancer
The power of combining cfDNA analysis with NGS to detect so- metastases from normal breast and 29 of 30 CRC metastases
matic mutations in cancer patients across multiple genes was from liver biopsies (Hao et al., 2017). However, conventional
first described in 2012 by Forshew et al. (2012). The approach, methylation profiling typically requires micrograms of DNA and
termed TAm-Seq, enabled analysis of nearly 6,000 genomic ba- bisulfite conversion, which destroy non-methylated DNA (Bhat-
ses at VAFs as low as 2%, greatly increasing the scope of ctDNA tacharjee et al., 2018), and so is not compatible with the limited
detection (Forshew et al., 2012). A similar approach to detect amounts of material typically associated with a liquid biopsy. The
tumor-specific chromosomal alterations was described by the potential of methylation profiling of cfDNA was recently demon-
Velculescu laboratory in the same year (Leary et al., 2012). The strated using a novel immunoprecipitation-based protocol that
clinical utility of cfDNA NGS approaches has also been demon- detected target cfDNA down to 0.001% (compared with 0.1%
strated in serial plasma samples to track genomic evolution of for a previously used hybridization detection approach) (Shen
metastatic cancers in response to therapy in advanced breast, et al., 2018). A similar approaching analyzing transcription start
ovarian, and lung cancers (Murtaza et al., 2013; Leary et al., sites where nucleosome occupancy results in different read
2012). To facilitate the use of ctDNA as a biomarker across depth coverage patterns for expressed and silent genes has
more tumor types and disease stages, the issues of false-posi- also been described (Ulz et al., 2016). This approach found anal-
tive (mutations being incorrectly called due to technical error ysis of cfDNA from metastatic patients could classify expressed
and noise) and false-negative (mutations being missed due to cancer driver genes in regions with somatic copy number gains
a lack of sensitivity of the assay) variant calling as well as with high accuracy. This has been further refined to infer acces-
CHIP, as described earlier, needs to be overcome. This has sibility of transcription factor binding sites from cell-free DNA
involved considerable efforts to increase the sensitivity of cfDNA fragmentation patterns that enables accurate prediction of tumor
NGS, including the addition of unique molecular identifiers (UMI) subtypes in prostate cancer (Ulz et al., 2019) with future potential
and digital error suppression (iDES) (Newman et al., 2014, 2016). for predictive biomarker development.
UMIs enable every cfDNA fragment to be uniquely identified and
a true consensus sequence of each molecule determined, NGS ctDNA Assays with Broadened Scope to Select
thereby reducing false-positive variant calling. The combination Targeted Therapies
of UMIs and iDES in the CAPPseq workflow correctly identified With the development of these high-sensitivity, high-specificity
EGFR mutations in 66 NSCLC samples with 92% sensitivity ctDNA assays, a broadened scope of ctDNA as a predictive
and 96% specificity, with detection of ctDNA down to 4 in 105 biomarker with clinical utility is being realized (Figure 1).
cfDNA molecules. A similar approach, TEC-Seq (targeted error Large-scale retrospective validation studies evaluating concor-
correction sequencing) also incorporating UMIs detected ctDNA dance with tumor mutations show promising results. As an
across a panel of 58 cancer-associated genes in 200 early-stage exemplar, a recent study of 10,593 patients showed >99.6%
cancer patients (Phallen et al., 2017) with mutations detected in technical success and 85.9% sensitivity using the Guardant360
59%–71% of stage I or II patients, depending on disease type. In cfDNA assay, which covers 73 cancer-related genes (Ode-
addition to the development of UMIs and improved bioinformatic gaard et al., 2018). This study also reported concordance be-
approaches, increased sensitivity of ctDNA detection has also tween cfDNA assay and tissue-based clinical genotyping
been described based on observations that ctDNA fragments across >750 patients with positive percentage agreement and
differ in length from cfDNA from healthy cells; typically 90– negative percentage agreement levels >99%. To move ctDNA
150 bp or >250 bp compared with 166 bp, respectively. These profiling into the clinical mainstream, prospective studies to
differences can be exploited both technically and bio- validate the clinical utility of NGS ctDNA assays are required.
informatically (Underhill et al., 2016; Mouliere et al., 2018; One such study is the TARGET study, (Rothwell et al., 2019)
Cristiano et al., 2019). Using an in vitro and in silico approach which aims to match patients with a broad range of advanced
to select fragments between 90 and 150 bp, Mouliere et al. cancers to early-phase clinical trials based on analysis of a
(2018) demonstrated a >2-fold median enrichment in detection 641 cancer-associated-gene cfDNA assay. Analysis of the first
of tumor DNA in >95% of 200 pan-cancer patients, where 100 patients showed that somatic mutation calling from cfDNA
analysis of size-selected cfDNA identified somatic mutations in could be performed, and data reported to a molecular tumor
samples that were otherwise undetectable. Physical selection board in a clinically feasible timescale showed good concor-
of low-frequency ctDNA is technically challenging, however in dance with tissue (78% concordance) and identified potentially
silico analysis of differences in fragmentation patterns from actionable mutations in 41% of patients (Rothwell et al., 2019).
shallow whole-genome sequencing (sWGS) also increased the Of these patients 11 of 41 (27%) went on to a match therapy,
sensitivity of ctDNA detection in 236 patients encompassing which reflects a similar level of recruitment to conventional tis-
seven cancer types with sensitivities from 57% to >99% and a sue-based targeted approaches and does not reflect a failing of
specificity of 98% (Cristiano et al., 2019). ctDNA analysis, rather the insufficiency of targeted therapy
In addition to improved mutational analysis, methylation drugs for detected and potentially targetable mutations (Tan-
profiling of cfDNA also has potential to increase ctDNA detection nock and Hickman, 2019).
sensitivity by encompassing tissue- and cancer-specific large- Minimal Residual Disease Monitoring and Anticipation
scale epigenetic alterations (Figure 1). As cancer methylome of Disease Recurrence Using ctDNA
mapping expands, DNA methylation profiles can differentiate tu- A more technically challenging approach using ctDNA is its
mor and normal tissues. Four common cancers were differenti- deployment as an early warning of relapsing disease after

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treatment with curative intent, which could direct adjuvant ther- Circulating Tumor Cells as Clinical Biomarkers and to
apy decision making or earlier intervention (Figure 1). The hy- Support Drug Development
pothesis is that ctDNA reflects tumor burden, and its level in- Although technical challenges remain for ctDNA assay develop-
creases as tumor regrowth occurs. The obvious advantage of ment, it has become the ‘‘go to’’ liquid biopsy in clinical trials,
the approach is the ease of longitudinal blood sample collection. with an avalanche of studies reporting encouraging data on its
One of the first studies to highlight the utility of ctDNA in minimal clinical utility as a predictive and response biomarker and an
residual disease (MRD) monitoring was reported by Nick Turn- MRD monitoring tool. In contrast, CTCs have only recently
er’s group following analysis of a prospective cohort of 55 gained substantial traction beyond their FDA-approved use a
early-stage breast cancer patients at high risk of relapse (Gar- decade ago as prognostic biomarkers in breast cancer, prostate
cia-Murillas et al., 2015). ctDNA detection at completion of treat- cancer, and CRC (Cristofanilli et al., 2004; Cohen et al., 2008; De
ment predicted metastatic relapse with high accuracy, and mu- Bono et al., 2008, Figure 1). The primary obstacle to further clin-
tation tracking in serial samples detected disease relapse ical implementation has been the high technical bar of rare and
7.9 months earlier than clinical surveillance (Garcia-Murillas phenotypically heterogeneous tumor cell detection, enrichment,
et al., 2015). More recently, the same group has shown that, in and isolation with typically only 1 CTC/mL of blood (Miller et al.,
a cohort of early-stage breast cancer patients, detection of 2010). Nevertheless, as CTC platforms evolve and improve (Pan-
ctDNA during follow-up was associated with relapse (hazard ra- tel and Alix- Panabieres, 2019), attaining the full potential of CTC
tio [HR], 25.2) and detection of ctDNA at diagnosis was also analyses, capturing DNA, RNA, and protein readouts, remains
associated with relapse-free survival (HR, 5.8). In this cohort, both tantalizing and pertinent as evidence mounts for therapy
ctDNA detection had a median lead time of 10.7 months (Gar- resistance mechanisms not readily measureable in ctDNA, for
cia-Murillas et al., 2019). Similar studies showing an association example, via phenotype switching. Moreover, while ctDNA con-
between disease-free survival and detection of ctDNA post centration is commonly associated with tumor burden (a balance
definitive treatment in numerous tumor types, including CRC, of tumor cell proliferation and death), mechanisms governing tu-
breast carcinoma, pancreatic carcinoma, and lung carcinoma, mor cell invasion, migration, and extra- and intravasation do not
have also been reported (reviewed in Coakley et al., 2019). As evoke an obligatory link to tumor size; small primary tumors can
discussed earlier, mutation panels can be tailored to each pa- metastasize, and large tumors do not always do so. The differing,
tient informed by sequencing their tumor with selection of driver, albeit incompletely understood, underlying mechanisms result-
truncal mutations for highly sensitive MRD monitoring. Tracking ing in ctDNA shedding and dissemination of CTCs emphasize
multiple mutations rather than a single point mutation also in- the complementarity of these distinct liquid biopsy approaches.
creases the sensitivity of ctDNA detection for MRD (Newman The robust and semi-automated CellSearch platform enriches
et al., 2014), an approach deployed in the TRACERx study of CTCs based on EpCAM expression and identifies these cells
NSCLC evolution (Abbosh et al., 2017). In this study, which fol- based on the absence of CD45 and the presence of cytokeratin
lows NSCLC patients after curative intent surgery, generation (CK) expression. CellSearch is considered the gold standard for
of patient-specific multiplexed PCR panels targeting a median CTC enrichment and enumeration and is FDA approved as a
of 18.5 or 28 mutations per patient in lung adenocarcinoma prognostic biomarker for breast cancer, prostate cancer, and
and squamous cell carcinoma patients, respectively, detected CRC (with a threshold of 5, 5, and 3 CTCs/7.5 mL of blood,
at least two SNVs in 13 of 14 (93%) patients prior to, or at, clinical respectively) (Cristofanilli et al., 2004; Cohen et al., 2008; De
relapse with a median interval between ctDNA detection and Bono et al., 2008). CellSearch CTCs are also prognostic for pro-
NSCLC relapse by computed tomography imaging of 70 days gression-free survival and overall survival in small-cell lung can-
(Abbosh et al., 2017). This approach has been further exempli- cer (SCLC) and NSCLC (50 and 5 CTCs/7.5 mL of blood, respec-
fied by the multiplexed targeted digital sequencing (TARDIS) tively) and can be used to monitor therapy response and relapse
approach, which analyzed up to 115 mutations per patient in, for example, lung, breast, and prostate cancers (Hou et al.,
from 33 women with stage I to III breast cancer, resulting in sen- 2009; Krebs et al., 2011; Bidard et al., 2014; Lorente et al.,
sitivities of 91% and 53% at VAFs of 3 in 104 and 3 in 105, 2016; Vogelzang et al., 2017; Heller et al., 2018; Pantel et al.,
respectively (McDonald et al., 2019). The sensitivities of these 2019). However, if epithelial-to-mesenchymal transition results
techniques and promising initial results suggest that personal- in downregulated EpCAM and/or CK expression, CTCs become
ized ctDNA tracking has great potential in the clinical manage- invisible to the CellSearch platform, and marker-independent
ment of patients treated with curative intent. platforms that capture epithelial and mesenchymal CTCs will
In summary, there is mounting support for the use of ctDNA as have better sensitivity as a result (Krebs et al., 2014). Notably,
a clinically applicable predictive biomarker of treatment although prognostic, CellSearch CTCs were detectable in the
response and resistance in cancer (Figure 1). There are peripheral blood of only 32% of 101 metastatic NSCLC patients
numerous approaches available, including targeted small-panel (Krebs et al., 2011). Several platforms for marker-independent
digital PCR, which enables quantitative measurement of limited CTC enrichment based on size and physical properties are under
numbers of mutations at sensitivities down to 0.001%, for a rela- evaluation (Krebs et al., 2014; Chudziak et al., 2016; Janning
tively low cost. The other extreme includes NGS-based broad- et al., 2019) but are not yet approved as clinical tests. CTC isola-
panel and whole-genome approaches such as methylation tion technologies (such as DEPArray@ or ALS@) can be imple-
profiling, which tend to be semi-quantitative, with poorer sensi- mented after CTC enrichment. However, any enrichment step
tivities and higher costs, but which have the potential to provide is likely to result in lost CTCs. The ‘‘no cell left behind’’ platform
much more information. The appropriate approach will depend from EPIC Sciences dispenses with the enrichment step and
on the clinical question being asked. captures all nucleated cells in a blood sample onto slides,

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supporting analysis of a broader range of CTC types as exempli- tures, that long-term passage on plastic is likely to result in irre-
fied in prostate cancer and NSCLC (Werner et al., 2015; McDa- versible adaptation and clonal outgrowth. CTCs have also been
niel et al., 2017; Boffa et al., 2017). Captured cells are interro- used to generate patient CTC Derived eXplant (CDX) models in
gated with multiplexed immunofluorescence assays (including mice, first shown for CTCs in breast cancer patients (Baccelli
exclusion and tumor markers) with proprietary algorithms to pre- et al., 2013) and subsequently in SCLC where CDX models
sent candidate tumor cells (Keomanee-Dizon et al., 2020), and reproduce the donor patient’s response to standard-of-care
physical picking of such cells for downstream genomic analysis chemotherapy (Hodgkinson et al., 2014; Drapkin et al., 2018,
can confirm tumor origin. While ctDNA is becoming a routine Figure 1). Longitudinal blood samples from SCLC patients
approach to assess tumor mutations and copy number changes, have been used to generate paired models representing the dis-
CTCs also offer analysis of protein expression (Figure 1) and sub- ease before and after development of resistance to therapy, al-
cellular localization along with single-cell analysis of tumor het- lowing resistance to be studied in a scenario where post-treat-
erogeneity (e.g., Carter et al., 2017; Chemi et al., 2019). ment tumor biopsies are rarely obtained (Drapkin et al., 2018;
Predicting Therapy Responses with CTCs Simpson et al., 2020). Most recently, single-cell RNA sequencing
The leading exemplar of an FDA-approved CTC predictive of SCLC CTCs and CDX cells demonstrated the diversity of
biomarker is the detection of the constitutively active ARV7 changes that accompany acquired chemoresistance (Allison
androgen receptor splice variant associated with prostate can- Stewart et al., 2020). Disaggregated CDX cells can be cultured
cer resistance to anti-hormonal drugs abiraterone and enzaluta- ex vivo for short periods of time, limiting drift while allowing func-
mide using the EPIC Sciences platform (Figure 1). Nuclear ARV7 tion-testing experimentation (Lallo et al., 2019). SCLC CDX
expression in patient CTCs predicts better outcome on taxane models are thus attractive to study treatment efficacy and resis-
and worse outcomes on anti-hormonals, validating clinical utility tance mechanisms to support drug and biomarker development
of this assay (Scher et al., 2018; Pantel et al., 2019). An explor- and predict treatment responses in a disease where both CTCs
atory biomarker example of CTC analysis in clinical trials (Hou et al., 2012) and ctDNA (Mohan et al., 2020) are prevalent
involved estrogen receptor expression in breast cancer CTCs facilitating liquid biopsies in clinical trials. However, the time
using CellSearch alongside analysis of mutational status, which required to generate and passage CDX cells in mice is currently
suggested existence of multiple mechanisms of resistance to not compatible with informing clinical decisions for individual
endocrine therapy, including incomplete estrogen receptor SCLC patients, and the immune-compromised mouse back-
downregulation and emergence of estrogen receptor mutations ground renders them unsuitable for immunotherapy testing.
(Paoletti et al., 2018a, 2018b). In addition, data from breast can- SCLC is a highly aggressive, early metastatic cancer where suc-
cer studies suggests that characterization of both CTCs and cessful generation of CDX models correlates with high CTC
ctDNA may provide a better early prediction of outcome (Paoletti numbers; CDX models are possible in NSCLC (e.g., Morrow
et al., 2018b). CTC assays for the immune checkpoint (IC) ligand et al., 2016) and in prostate cancer (Tayoun et al., 2019), but
PD-L1 have also been developed using the EPIC Sciences, Cell have not yet been reported in multiple tumor types.
Search, and other platforms to explore their potential as predic- Predicting Risk of Disease Recurrence with CTCs
tive biomarkers for IC targeted therapies (Mazel et al., 2015; The presence of CTCs after treatment with curative intent might
Boffa et al., 2017; Guibert et al., 2018; Yue et al., 2018; Janning reflect the presence of micro-metastasis and portend a high risk
et al., 2019, Figure 1). of disease relapse. Several breast cancer studies provide evi-
As a proof-of-principle example of genomic analysis of CTCs, dence for this. A meta-analysis of more than 1,500 patients
a copy number alteration (CNA) signature predictive at baseline with non-metastatic breast cancer reported that CTCs were
of subsequent chemosensitivity was reported for SCLC; this detectable in 25% of patients prior to neoadjuvant therapy and
CTC CNA classifier correctly assigned 83% of 31 cases as che- associated with worse overall survival, disease-free survival,
mosensitive or chemorefractory (Carter et al., 2017). Clearly, a and recurrence-free survival but notably not with pathological
larger study is required to validate the CTC classifier, and its clin- complete response (Bidard et al., 2018). In the phase III SUC-
ical implementation will be warranted only when improved ther- CESS trial in high risk non-metastatic breast cancer patients,
apies beyond platinum-based chemotherapy are developed that detection of CTCs 2 years post adjuvant chemotherapy was
improve outcomes for chemorefractory SCLC patients. Howev- associated with worse outcome (Trapp et al., 2019), and in local-
er, this approach illustrates the potential for molecular character- ized breast cancer, the presence of CTCs at 5 years after diag-
ization of CTCs to provide predictive biomarkers that inform nosis occurred in 26 of 547 (4.8%) of patients and was associ-
treatment decisions as well as mechanistic insight into drug ated with a higher risk of late recurrence among patients with
sensitivity and resistance. hormone receptor-positive breast cancer (Sparano et al., 2018).
Several groups have demonstrated that CTCs can be cultured, The hypothesis that CTCs are associated with a high risk of
notably from breast and prostate cancer patients (e.g., Zhang relapse was further tested in stage I–IIIa patients with NSCLC un-
et al., 2013; Yu et al., 2014; Kolostova et al., 2014), with potential dergoing surgery with curative intent. CellSearch CTCs were un-
for real-time drug testing to predict donor patient responses with detectable in the peripheral blood samples in most patients, but
robust assays tailored to low cell numbers (Figure 1). CTC cell EpCAM- and CK-positive cells were present in the pulmonary
lines can also be derived, notably from patients with breast, colo- vein (PV) draining the cancerous lung (Crosbie et al., 2016;
rectal, and gastric cancers, and with SCLC (Yu et al., 2014; Rath Chemi et al., 2019) immediately prior to tumor resection in a sub-
et al., 2019, Brungs et al., 2020; Soler et al., 2018; Klameth et al., set of patients. Greater CellSearch CTC abundance in PV versus
2017) adding new tools for drug development and to explore the peripheral blood is likely to be attributable to proximity to the pri-
biology of advanced cancers, with the caveat, as with all cell cul- mary tumor and the fact that PV blood is drawn upstream of

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Review

capillary bed filtering. These PV circulating epithelial cells pre- through analysis of T cell subsets in a peripheral blood sample,
dicted the risk of lung-specific disease relapse in 100 patients using approaches such as T cell receptor sequencing. Consis-
enrolled on the TRACERx study (Chemi et al., 2019). A larger pro- tent with this are recent reports on blood-based early response
spective study is required to validate this finding, but enumera- biomarkers for IC blockade in melanoma patients. Valpione
tion of these epithelial cells in the PV at surgery may inform on et al. (2020) reported increases in productive T cell receptor se-
which patients should receive more frequent surveillance with quences in cfDNA and the expansion of a subset of cytotoxic
ctDNA and imaging after surgery. Of note, in a case study memory effector T cells. Fairfax et al. (2020) reported that a
when CellSearch PV CTCs were isolated and sequenced, these higher number of large T cell clones are detected in peripheral
early disseminating CTCs had a higher mutational overlap with blood from responding patients. Together, analysis of ctDNA
metastasis occurring 10 months later than did the resected pri- and T cell expansion represent promising developments in the
mary tumor, suggesting early dissemination of PV CTCs is hunt for liquid biopsies to inform the use of immunotherapies.
responsible for disease relapse (Chemi et al., 2019). This illus- CTC enumeration has not yet been explored as a predictive
trates the potential for CTCs to provide a better representation and/or response marker for IC therapy, but assays are available
of tumor heterogeneity than a tumor biopsy sample. It also sug- for analysis of PD-L1 expression in CTCs (Mazel et al., 2015;
gests that CTCs are enriched for the most aggressive cancer Boffa et al., 2017; Guibert et al., 2018; Yue et al., 2018; Janning
cells more likely to form metastasis and hence very relevant for et al., 2019) and their prognostic and predictive value is under
therapeutic intervention in advanced stage disease. This hypoth- investigation.
esis is further supported by molecular profiling of SCLC patient
CTCs revealing increased heterogeneity on the development of Conclusion
resistance to therapy (Allison Stewart et al., 2020). The field of liquid biopsies is rapidly evolving. Most notably,
ctDNA-based biomarkers to predict and monitor tumor re-
Liquid Biopsy to Guide Immunotherapy sponses to treatment and to monitor MRD have generated
Immunotherapy has transformed cancer therapy in several tu- enormous interest; research publications continue to demon-
mor types and patient subsets, with impressive durable re- strate the feasibility of ctDNA testing in a broad range of tumor
sponses most notably in melanoma, renal cancer, and NSCLC types (Figure 1). Numerous diagnostic companies now offer
(Kruger et al., 2019). Several candidate tumor-based predictive liquid biopsy testing, and drug developers are increasingly
biomarkers have emerged for IC therapy. Microsatellite insta- developing ctDNA companion diagnostics for their targeted
bility (MSI) results in a high number of gene mutations within tu- therapies. As a result, many oncologists are embracing the
mors and is the first pan-cancer indication approved for IC ease of blood sample collection and rapid turnaround time of
blockade (Le et al., 2017). Across several studies, TMB has ctDNA test results, adding this approach to assist in their treat-
shown predictive value for IC inhibition, and the utility of a ment decision making. Tumor biopsies, for the most part,
blood-based TMB measurement using ctDNA is under evalua- remain the gold standard, but as the sensitivity and specificity
tion (Gandara et al., 2018; Wang et al., 2019; Peters et al., of liquid biopsy assays continue to improve and real-world ev-
2019; Snyder et al., 2019). A retrospective analysis of the POP- idence continues to mount, it would seem that ctDNA-based
LAR and OAK clinical studies of the IC inhibitor atezolizumab liquid biopsies are heading toward mainstream in precision
versus chemotherapy in NSCLC showed an association of oncology. The technical challenges of CTCs remain, and
ctDNA TMB with clinical outcome, with a TMB >16 mut/Mb development of robust CTC assays that capture the required
demonstrating a median overall survival of 13.5 months for ate- phenotypic diversity takes a considerable time. However, in in-
zolizumab versus 6.8 months with docetaxel in the OAK study stances where the genome alone cannot predict treatment
(Gandara et al., 2018). Although the MYSTIC study of IC inhibi- response and downstream information, e.g., on drug-target
tors durvalumab plus tremelimumab in first-line NSCLC failed hitting, is informative, a robust CTC assay, such as the AR
to meet its primary endpoint, improved benefit for the immuno- variant in prostate cancer, comes to the fore (Figure 1). Given
therapy combination versus chemotherapy was observed at the relative ease of ctDNA assay development, CTC assay de-
higher blood-based TMB cut-offs (Peters et al., 2019). Assays velopers should perhaps double down on what ctDNA cannot
for measurement of MSI in ctDNA have also been reported achieve in the biomarker space. The common problem of tar-
(Georgiadis et al., 2019; Willis et al., 2019), raising the opportu- geted therapy resistance makes continuous monitoring of a
nity for analysis of both TMB and MSI from a blood sample to patient’s treatment response mission critical, and here liquid
predict clinical benefit from IC therapy. Studies also indicate biopsies will play a significant role, anticipating when to switch
that changes in ctDNA can provide an early biomarker of treatment (Figure 1). The liquid biopsy sensitivity challenges of
response to IC therapy and in long-term responders, monitoring MRD monitoring are beginning to be met, and a combined pre-
of ctDNA can detect MRD and predict relapse (Hellmann et al., treatment tumor biopsy with subsequent serial liquid biopsies
2020; Goldberg et al., 2018; Anagnostou et al., 2019; Moding based on each patient’s tumor profile form the basis of trials
et al., 2020). to test whether patient benefit follows earlier intervention
Recently, it was shown that expansion of T cell clones within based on ctDNA warning of relapse after curative intent treat-
the tumor is paralleled by their expansion within the peripheral ment. The biomarker promise of TEPs, tumor exosomes, and
blood, and patients with such T cell clonotypic expansion circulating RNAs have yet to be realized in the clinic, but
respond better to PD1 checkpoint blockade (Wu et al., 2020). ongoing translational research raises expectations that new
This new finding represents an important observation, high- liquid biopsies are on the horizon, adding to the battery of
lighting the potential for detection of responding patients blood tests to support future precision oncology.

Cancer Cell 37, April 13, 2020 491

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Review

The next frontier for liquid biopsies is perhaps to answer the Baccelli, I., Schneeweiss, A., Riethdorf, S., Stenzinger, A., Schillert, A., Vogel,
€uerle, T., Wallwiener, M., et al. (2013). Identification of
V., Klein, C., Saini, M., Ba
question of whether or not they might assist with the pressing a population of blood circulating tumor cells from breast cancer patients that
need for predictive biomarkers for immunotherapy—or is that a initiates metastasis in a xenograft assay. Nat. Biotechnol. 31, 539–544.
step too far, requiring an in-depth understanding of the tumor mi-
Bettegowda, C., Sausen, M., Leary, R.J., Kinde, I., Wang, Y., Agrawal, N., Bar-
cro-environment and thus a mandatory tumor biopsy? TMB in tlett, B.R., Wang, H., Luber, B., Alani, R.M., et al. (2014). Detection of circu-
ctDNA, IC protein in CTCs (Figure 1), receptor sequencing of pe- lating tumor DNA in early- and late-stage human malignancies. Sci. Transl.
ripheral T cell subsets are all in play; their clinical utility remains to Med. 6, 224ra24.

be defined, but the recent developments suggesting liquid bi- Bhattacharjee, R., Moriam, S., Umer, M., Nguyen, N.T., and Shiddiky, M.J.A.
opsies could be developed to support immunotherapy decision (2018). DNA methylation detection: recent developments in bisulfite free elec-
trochemical and optical approaches. Analyst 143, 4802–4818.
making are an exciting ‘‘watch this space.’’ The application of
liquid biopsies for early detection of cancers is a topic of huge in- Bidard, F.-C., Peeters, D.J., Fehm, T., Nolé, F., Gisbert-Criado, R., Mavroudis,
terest and importance; while many of the issues discussed here D., Grisanti, S., Generali, D., Garcia-Saenz, J.A., Stebbing, J., et al. (2014).
Clinical validity of circulating tumour cells in patients with metastatic breast
that touch upon sensitivity and specificity of liquid biopsies are cancer: a pooled analysis of individual patient data. Lancet Oncol. 15,
particularly germane, early detection is beyond the remit of this 406–414.
review.
Bidard, F.C., Michiels, S., Riethdorf, S., Mueller, V., Esserman, L.J., Lucci, A.,
The universal replacement of tumor biopsies with liquid bi- Naume, B., Horiguchi, J., Gisbert-Criado, R., Sleijfer, S., et al. (2018). Circu-
opsies seems unrealistic; however, as ctDNA, CTC, and the lating tumor cells in breast cancer patients treated by neoadjuvant chemo-
therapy: a meta-analysis. J. Natl. Cancer Inst. 110, 560–567.
additional aforementioned blood tests improve, it seems likely
that they will become an increasingly used tool for cancer patient Boffa, D.J., Graf, R.P., Salazar, M.C., Hoag, J., Lu, D., Krupa, R., Louw, J., Du-
management, especially when serial assessment of patients is gan, L., Wang, Y., Landers, M., et al. (2017). Cellular expression of PD-L1 in the
peripheral blood of lung cancer patients is associated with worse survival.
required and/or an invasive tumor biopsy is not feasible. Cancer Epidemiol. Biomarkers Prev. 26, 1139–1145.

De Bono, J.S., Scher, H.I., Montgomery, R.B., Parker, C., Miller, M.C., Tissing,
ACKNOWLEDGMENTS H., Doyle, G.V., Terstappen, L.W., Pienta, K.J., and Raghavan, D. (2008).
Circulating tumor cells predict survival benefit from treatment in metastatic
E.K., D.R., G.B., and C.D. are funded by Core Funding to CRUK Manchester castration-resistant prostate cancer. Clin. Cancer Res. 14, 6302–6309.
Institute, United Kingdom (A27412); and the Manchester CRUK Centre Award,
Bronkhorst, A.J., Ungerer, V., and Holdenrieder, S. (2019). The emerging role
United Kingdom (A25254). Support was received from the CRUK Manchester
of cell-free DNA as a molecular marker for cancer management. Biomol.
Experimental Cancer Medicines Centre, United Kingdom (A25146); the CRUK Detect. Quantif. 17, 100087.
Lung Cancer Centre of Excellence, United Kingdom (A29420); and the Man-
chester NIHR Biomedical Research Centre, United Kingdom. We thank Ekram Brungs, D., Minaei, E., Piper, A.-K., Perry, J., Splitt, A., Carolan, M., Ryan, S.,
Aidaros-Talbot for assistance with this manuscript. Declaration of Interests Wu, X.J., Corde, S., Tehei, M., et al. (2020). Establishment of novel long-term
Research funding has been received by C.D. from AstraZeneca, United cultures from EpCAM positive and negative circulating tumour cells from pa-
Kingdom; Astex Pharmaceuticals, United States; Bioven, United Kindom; tients with metastatic gastroesophageal cancer. Sci. Rep. 10, 539.
Amgen, United States; Carrick Therapeutics, Republic of Ireland; Merck AG,
Germany; Taiho Oncology, Japan; Biolidics Limited, Singapore; Angle PLC, Carter, L., Rothwell, D.G., Mesquita, B., Smowton, C., Leong, H.S., Fernan-
United Kingdom; Menarini Diagnostics, Italy; GSK, United Kingdom; Bayer, dez-Gutierrez, F., Li, Y., Burt, D.J., Antonello, J., Morrow, C.J., et al. (2017).
Germany; Boehringer Ingelheim, Germany; Roche, Switzerland; BMS, United Molecular analysis of circulating tumor cells identifies distinct copy-number
States; Novartis, Switzerland; Celgene, United States; Thermo Fisher, United profiles in patients with chemosensitive and chemorefractory small-cell lung
States. C.D. is on the advisory board and has received consultancy fees/ cancer. Nat. Med. 23, 114–119.
honoraria from AstraZeneca, United Kingdom and Biocartis, Belgium.
Chemi, F., Rothwell, D.G., McGranahan, N., Gulati, S., Abbosh, C., Pearce,
S.P., Zhou, C., Wilson, G.A., Jamal-Hanjani, M., Birkbak, N., et al. (2019). Pul-
monary venous circulating tumor cell dissemination before tumor resection
DECLARATION OF INTERESTS and disease relapse. Nat. Med. 25, 1534–1539.

Research funding has been received by C.D. from AstraZeneca, Astex Phar- Chudziak, J., Burt, D.J., Mohan, S., Rothwell, D.G., Mesquita, B., Antonello, J.,
maceuticals, Bioven, Amgen, Carrick Therapeutics, Merck AG, Taiho Dalby, S., Ayub, M., Priest, L., Carter, L., et al. (2016). Clinical evaluation of a
Oncology, Clearbridge Biomedics, Angle PLC, Menarini Diagnostics, GSK, novel microfluidic device for epitope-independent enrichment of circulating
Bayer, Boehringer Ingelheim, Roche, BMS, Novartis, Celgene, Thermo Fisher. tumour cells in patients with small cell lung cancer. Analyst 141, 669–678.
C.D. is on the advisory board and has received consultancy fees/honoraria
from AstraZeneca and Biocartis. Coakley, M., Garcia-Murillas, I., and Turner, N.C. (2019). Molecular residual
disease and adjuvant trial design in solid tumors. Clin. Cancer Res. 25,
6026–6034.
REFERENCES
Cohen, S.J., Punt, C.J., Iannotti, N., Saidman, B.H., Sabbath, K.D., Gabrail,
N.Y., Picus, J., Morse, M., Mitchell, E., Miller, M.C., et al. (2008). Relationship
Abbosh, C., Birkbak, N.J., Wilson, G.A., Jamal-Hanjani, M., Constantin, T., of circulating tumor cells to tumor response, progression-free survival and
Salari, R., Le Quesne, J., Moore, D.A., Veeriah, S., Rosenthal, R., et al. overall survival in patients with metastatic colorectal cancer. J. Clin. Oncol.
(2017). Phylogenetic ctDNA analysis depicts early-stage lung cancer evolu- 26, 3213–3221.
tion. Nature 545, 446–451.
Cristiano, S., Leal, A., Phallen, J., Fiksel, J., Adleff, V., Bruhm, D.C., Jensen,
Allison Stewart, C., Gay, C.M., Xi, Y., Sivajothi, S., Sivakamasundari, V., Fuji- S.Ø., Medina, J.E., Hruban, C., White, J.R., et al. (2019). Genome-wide cell-
moto, J., Bolisetty, M., Hartsfield, P.M., Balasubramaniyan, V., Chalishazar, free DNA fragmentation in patients with cancer. Nature 570, 385–389.
M.D., et al. (2020). Single-cell analyses reveal increased intratumoral hetero-
geneity after the onset of therapy resistance in small-cell lung cancer. Nat. Cristofanilli, M., Budd, G.T., Ellis, M.J., Stopeck, A., Matera, J., Miller, M.C.,
Cancer. https://doi.org/10.1038/s43018-019-0020-z. Reuben, J.M., Doyle, G.V., Allard, W.J., Terstappen, L.W., and Hayes, D.F.
(2004). Circulating tumor cells, disease progression, and survival in metastatic
Anagnostou, V., Forde, P.M., White, J.R., Niknafs, N., Hruban, C., Naidoo, J., breast cancer. N. Engl. J. Med. 351, 781–791.
Marrone, K., Sivakumar, I.K.A., Bruhm, D.C., Rosner, S., et al. (2019). Dy-
namics of tumor and immune responses during immune checkpoint blockade Crosbie, P.A.J., Shah, R., Krysiak, P., Zhou, C., Morris, K., Tugwood, J.,
in non-small cell lung cancer. Cancer Res. 79, 1214–1225. Booton, R., Blackhall, F., and Dive, C. (2016). Circulating tumor cells detected

492 Cancer Cell 37, April 13, 2020

Cancer Cell

Review
in the tumor-draining pulmonary vein are associated with disease recurrence €nne, P.A., Yang, J.C.-H., Kim, D.-W., Planchard, D., Ohe, Y., Ramalingam,
Ja
after surgical resection of NSCLC. J. Thorac. Oncol. 11, 1793–1797. S.S., Ahn, M.-J., Kim, S.-W., Su, W.-C., Horn, L., et al. (2015). AZD9291 in
EGFR inhibitor-resistant non-small-cell lung cancer. N. Engl. J. Med. 372,
Drapkin, B.J., George, J., Christensen, C.L., Mino-Kenudson, M., Dries, R., 1689–1699.
Sundaresan, T., Phat, S., Myers, D.T., Zhong, J., Igo, P., et al. (2018). Genomic
and functional fidelity of small cell lung cancer patient-derived xenografts. Janning, M., Kobus, F., Babayan, A., Wikman, H., Velthaus, J.-L., Bergmann,
Cancer Discov. 8, 600–615. S., Schatz, S., Falk, M., Berger, L.-A., and Böttcher, L.-M. (2019). Determina-
tion of PD-L1 expression in circulating tumor cells of NSCLC patients and cor-
Fairfax, B.P., Taylor, C.A., Watson, R.A., Nassiri, I., Danielli, S., Fang, H., Mahé, relation with response to PD-1/PD-L1 inhibitors. Cancers (Basel) 11, https://
E.A., Cooper, R., Woodcock, V., Traill, Z., et al. (2020). Peripheral CD8+ T cell doi.org/10.3390/cancers11060835.
characteristics associated with durable responses to immune checkpoint
blockade in patients with metastatic melanoma. Nat. Med. 26, 193–199. Jenkins, S., Yang, J.C.-H., Ramalingam, S.S., Yu, K., Patel, S., Weston, S.,
€nne, P.A., Mitsudomi, T., and Goss, G.D. (2017).
Hodge, R., Cantarini, M., Ja
Forshew, T., Murtaza, M., Parkinson, C., Gale, D., Tsui, D.W., Kaper, F., Daw- Plasma ctDNA analysis for detection of the EGFR T790M mutation in patients
son, S.J., Piskorz, A.M., Jimenez-Linan, M., Bentley, D., et al. (2012). Noninva- with advanced non–small cell lung cancer. J. Thorac. Oncol. 12, 1061–1070.
sive identification and monitoring of cancer mutations by targeted deep
sequencing of plasma DNA. Sci. Transl. Med. 4, 136ra68. Kamisawa, T., Wood, L.D., Itoi, T., and Takaori, K. (2016). Pancreatic cancer.
Lancet 388, 73–85.
Gandara, D.R., Paul, S.M., Kowanetz, M., Schleifman, E., Zou, W., Li, Y., Ritt-
Keomanee-Dizon, K., Shishido, S.N., and Kuhn, P. (2020). Circulating tumor
meyer, A., Fehrenbacher, L., Otto, G., Malboeuf, C., et al. (2018). Blood-based
cells: high-throughput imaging of CTCs and bioinformatic analysis. Recent Re-
tumor mutational burden as a predictor of clinical benefit in non-small-cell lung
sults Cancer Res. 215, 89–104.
cancer patients treated with atezolizumab. Nat. Med. 24, 1441–1448.
Klameth, L., Rath, B., Hochmaier, M., Moser, D., Redl, M., Mungenast, F.,
Garcia-Murillas, I., Schiavon, G., Weigelt, B., Ng, C., Hrebien, S., Cutts, R.J.,
Gelles, K., Ulsperger, E., Zeillinger, R., and Hamilton, G. (2017). Small cell
Cheang, M., Osin, P., Nerurkar, A., Kozarewa, I., et al. (2015). Mutation tracking
lung cancer: model of circulating tumor cell tumorospheres in chemoresist-
in circulating tumor DNA predicts relapse in early breast cancer. Sci. Transl.
ance. Sci. Rep. 7, 5337.
Med. 7, 302ra133.
Kolostova, K., Cegan, M., and Bobek, V. (2014). Circulating tumour cells in pa-
Garcia-Murillas, I., Chopra, N., Comino-Méndez, I., Beaney, M., Tovey, H., tients with urothelial tumours: enrichment and in vitro culture. Can. Urol. As-
Cutts, R.J., Swift, C., Kriplani, D., Afentakis, M., Hrebien, S., et al. (2019). soc. J. 8, E715–E720.
Assessment of molecular relapse detection in early-stage breast cancer.
JAMA Oncol. https://doi.org/10.1001/jamaoncol.2019.1838. Krebs, M.G., Sloane, R., Priest, L., Lancashire, L., Hou, J.M., Greystoke, A.,
Ward, T.H., Ferraldeschi, R., Hughes, A., Clack, G., et al. (2011). Evaluation
Georgiadis, A., Durham, J.N., Keefer, L.A., Bartlett, B.R., Zielonka, M., Mur- and prognostic significance of circulating tumor cells in patients with non-
phy, D., White, J.R., Lu, S., Verner, E.L., Ruan, F., et al. (2019). Noninvasive small-cell lung cancer. J. Clin. Oncol. 29, 1556–1563.
detection of microsatellite instability and high tumor mutation burden in cancer
patients treated with PD-1 blockade. Clin. Cancer Res. 25, 7024–7034. Krebs, M.G., Metcalf, R.L., Carter, L., Brady, G., Blackhall, F.H., and Dive, C.
(2014). Molecular analysis of circulating tumour cells – biology and biomarkers.
Goldberg, S.B., Narayan, A., Kole, A.J., Decker, R.H., Teysir, J., Carriero, N.J., Nat. Rev. 11, 129–144.
Lee, A., Nemati, R., Nath, S.K., Mane, S.M., et al. (2018). Early assessment of
lung cancer immunotherapy response via circulating tumor DNA. Clin. Cancer Kruger, S., Ilmer, M., Kobold, S., Cadilha, B.L., Endres, S., Ormanns, S.,
Res. 24, 1872–1880. Schuebbe, G., Renz, B.W., D’Haese, J.G., Schloesser, H., et al. (2019). Ad-
vances in cancer immunotherapy 2019 - latest trends. J. Exp. Clin. Cancer
Guibert, N., Delaunay, M., Lusque, A., Boubekeur, N., Rouquette, I., Clermont, Res. 38, 268.
E., Mourlanette, J., Gouin, S., Dormoy, I., Favre, G., et al. (2018). PD-L1
expression in circulating tumor cells of advanced non-small cell lung cancer Kwapisz, D. (2017). The first liquid biopsy test approved. Is it a new era of mu-
patients treated with nivolumab. Lung Cancer 120, 108–112. tation testing for non-small cell lung cancer? Ann. Transl. Med. 5, 46.

Hao, X., Luo, H., Krawczyk, M., Wei, W., Wang, W., Wang, J., Flagg, K., Hou, Lallo, A., Gulati, S., Schenk, M.W., Khandelwal, G., Berglund, U.W., Pateras,
J., Zhang, H., Yi, S., et al. (2017). DNA methylation markers for diagnosis and I.S., Chester, C.P.E., Pham, T.M., Kalderen, C., Frese, K.K., et al. (2019).
prognosis of common cancers. Proc. Natl. Acad. Sci. U S A 114, 7414–7419. Ex vivo culture of cells derived from circulating tumour cell xenograft to sup-
port small cell lung cancer research and experimental therapeutics. Br. J.
Heitzer, E., Haque, I.S., Roberts, C.E.S., and Speicher, M.R. (2019). Current Pharmacol. 176, 436–450.
and future perspectives of liquid biopsies in genomics-driven oncology. Nat.
Rev. Genet. 20, 71–88. Le, D.T., Durham, J.N., Smith, K.N., Wang, H., Bartlett, B.R., Aulakh, L.K., Lu,
S., Kemberling, H., Wilt, C., Luber, B.S., et al. (2017). Mismatch-repair defi-
Heller, G., McCormack, R., Kheoh, T., Molina, A., Smith, M.R., Dreicer, R., ciency predicts response of solid tumors to PD-1 blockade. Science 357,
Saad, F., de Wit, R., Aftab, D.T., Hirmand, M., et al. (2018). Circulating tumor 409–413.
cell number as a response measure of prolonged survival for metastatic
Leary, R.J., Sausen, M., Kinde, I., Papadopoulos, N., Carpten, J.D., Craig, D.,
castration-resistant prostate cancer: a comparison with prostate-specific an-
O’Shaughnessy, J., Kinzler, K.W., Parmigiani, G., Vogelstein, B., et al. (2012).
tigen across five randomized phase III clinical trials. J. Clin. Oncol. 36,
Detection of chromosomal alterations in the circulation of cancer patients with
572–580.
whole-genome sequencing. Sci. Transl. Med. 4, 162ra154.
Hellmann, M.D., Nabet, B.Y., Rizvi, H., Chaudhuri, A.A., Wells, D.K., Dunphy,
Leon, S.A., Shapiro, B., Sklaroff, D.M., and Yaros, M.J. (1977). Free DNA in the
M.P., Chabon, J.J., Liu, C.L., Hui, A.B., Arbour, K.C., et al. (2020). Circulating
serum of cancer patients and the effect of therapy. Cancer Res. 37, 646–650.
tumor DNA analysis to assess risk of progression after long-term response to
PD-(L)1 blockade in NSCLC. Clin. Cancer Res. https://doi.org/10.1158/1078- Liu, J., Chen, X., Wang, J., Zhou, S., Wang, C.L., Ye, M.Z., Wang, X.Y., Song,
0432.CCR-19-3418. Y., Wang, Y.Q., Zhang, L.T., et al. (2019). Biological background of the
genomic variations of cf-DNA in healthy individuals. Ann. Oncol. 30, 464–470.
Herbst, R.S., Morgensztern, D., and Boshoff, C. (2018). The biology and man-
agement of non-small cell lung cancer. Nature 553, 446–454. Lo, Y.M., Corbetta, N., Chamberlain, P.F., Rai, V., Sargent, I.L., Redman, C.W.,
and Wainscoat, J.S. (1997). Presence of fetal DNA in maternal plasma and
Hodgkinson, C.L., Morrow, C.J., Li, Y., Metcalf, R.L., Rothwell, D.G., Trapani, serum. Lancet 350, 485–487.
F., Polanski, R., Burt, D.J., Simpson, K.L., Morris, K., et al. (2014). Tumorige-
nicity and genetic profiling of circulating tumor cells in small-cell lung cancer. Lorente, D., Olmosb, D., Mateoa, J., Bianchinia, D., Seeda, G., Fleisher, M.,
Nat. Med. 20, 897–903. Danilac, D.C., Flohra, P., Crespoa, M., Figueiredoa, I., et al. (2016). Decline
in circulating tumor cell count and treatment outcome in advanced prostate
Hou, J.-M., Greystoke, A., Lancashire, L., Cummings, J., Ward, T., Board, R., cancer. Eur. Urol. 70, 985–992.
Amir, E., Hughes, S., Krebs, M., Hughes, A., et al. (2009). Evaluation of circu-
lating tumor cells and serological cell death biomarkers in small cell lung can- Mandel, P., and Metais, P. (1948). Les acides nucléiques du plasma sanguin
cer patients undergoing chemotherapy. Am. J. Pathol. 175, 808–816. chez l’homme. C. R. Seances Soc. Biol. Fil. 142, 241–243.

Cancer Cell 37, April 13, 2020 493

 Cancer Cell

Review
Mazel, M., Jacot, W., Pantel, K., Bartkowiak, K., Topart, D., Cayrefourcq, L., mor cells documents heterogenous resistance mechanisms. Cancer Res.
Rossille, D., Maudelonde, T., Fest, T., and Alix-Panabières, C. (2015). Frequent 78, 1110–1122.
expression of PD-L1 on circulating breast cancer cells. Mol. Oncol. 9,
1773–1782. Paoletti, C., Schiavon, G., Dolce, E.M., Darga, E.P., Carr, T.H., Geradts, J.,
Hoch, M., Klinowska, T., Lindemann, J., Marshall, G., et al. (2018b). Circulating
McDaniel, A.S., Ferraldeschi, R., Krupa, R., Landers, M., Graf, R., Louw, J., biomarkers and resistance to endocrine therapy in metastatic breast cancers:
Jendrisak, A., Bales, N., Marrinucci, D., Zafeiriou, Z., et al. (2017). Phenotypic correlative results from AZD9496 oral SERD phase I trial. Clin. Cancer Res. 24,
diversity of circulating tumour cells in patients with metastatic castration-resis- 5860–5872.
tant prostate cancer. BJU Int. 120, E30–E44.
Parseghian, C.M., Loree, J.M., Morris, V.K., Liu, X., Clifton, K.K., Napolitano,
McDonald, B.R., Contente-Cuomo, T., Sammut, S.J., Odenheimer-Bergman, S., Henry, J.T., Pereira, A.A., Vilar, E., Johnson, B., et al. (2019). Anti-EGFR-
A., Ernst, B., Perdigones, N., Chin, S.F., Farooq, M., Mejia, R., Cronin, P.A., resistant clones decay exponentially after progression: implications for anti-
et al. (2019). Personalized circulating tumor DNA analysis to detect residual EGFR re-challenge. Ann. Oncol. 30, 243–249.
disease after neoadjuvant therapy in breast cancer. Sci. Transl. Med. 11,
https://doi.org/10.1126/scitranslmed.aax7392. Peters S., Cho B.C., Reinmuth N., Lee K.H., Luft A., Ahn M.-J., Baas P., Dols,
M.C., Smolin, A., Vicente, D., et al. (2019). CT074: Tumor mutational burden
Miller, C.M., Doyle, G.V., and Terstappen, L.W. (2010). Significance of circu- (TMB) as a biomarker of survival in metastatic non-small cell lung cancer
lating tumor cells detected by the CellSearch system in patients with metasta- (mNSCLC): blood and tissue TMB analysis from MYSTIC, a phase III study
tic breast colorectal and prostate cancer. J. Oncol. 2010, 617421. of first-line durvalumab ± tremelimumab vs chemotherapy [abstract]. In: Pro-
ceedings of the American Association for Cancer Research Annual Meeting
Moding, E.J., Liu, Y., Nabet, B.Y., Chabon, J.J., Chaudhuri, A.A., Hui, A.B., Bo- 2019; 2019 Mar 29–Apr 3; Atlanta, GA. Philadelphia (PA): AACR. Abstract
nilla, R.F., Ko, R.B., Yoo, C.H., Gojenola, L., et al. (2020). Circulating tumor no. CT074.
DNA dynamics predict benefit from consolidation immunotherapy in locally
advanced non-small-cell lung cancer. Nat. Cancer 1, 176–183. Phallen, J., Sausen, M., Adleff, V., Leal, A., Hruban, C., White, J., Anagnostou,
V., Fiksel, J., Cristiano, S., Papp, E., et al. (2017). Direct detection of early-
Mohan, S., Foy, V., Ayub, M., Leong, H.S., Schofield, P., Sahoo, S., Des- stage cancers using circulating tumor DNA. Sci. Transl. Med. 9, https://doi.
camps, T., Kilerci, B., Smith, N.K., Carter, M., et al. (2020). Profiling of circu- org/10.1126/scitranslmed.aan2415.
lating free DNA using targeted and genome-wide sequencing in patients
with SCLC. J. Thorac. Oncol. 15, 216–230. Rath, B., Klameth, L., Plangger, A., Hochmair, M., Ulsperger, E., Huk, I., Zeil-
linger, R., and Hamilton, G. (2019). Expression of proteolytic enzymes by small
Morrow, C.J., Trapani, F., Metcalf, R.L., Bertolini, G., Hodgkinson, C.L., Khan- cell lung cancer circulating tumor cell lines. Cancers (Basel) 11, 114.
delwal, G., Kelly, P., Galvin, M., Carter, L., Simpson, K.L., et al. (2016). Tu-
mourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: Razavi, P., Li, B.T., Brown, D.N., Jung, B., Hubbell, E., Shen, R., Abida, W., Ju-
a clinical case study. Ann. Oncol. 27, 1155–1160. luru, K., De Bruijn, I., Hou, C., et al. (2019). High-intensity sequencing reveals
the sources of plasma circulating cell-free DNA variants. Nat. Med. 25,
Mouliere, F., Chandrananda, D., Piskorz, A.M., Moore, E.K., Morris, J., Ahl- 1928–1937.
born, L.B., Mair, R., Goranova, T., Marass, F., Heider, K., et al. (2018).
Enhanced detection of circulating tumor DNA by fragment size analysis. Sci. Rothwell, D.G., Ayub, M., Cook, N., Thistlethwaite, F., Carter, L., Dean, E.,
Transl. Med. 10, https://doi.org/10.1126/scitranslmed.aat4921. Smith, N., Villa, S., Dransfield, J., Clipson, A., et al. (2019). Utility of ctDNA to
support patient selection for early phase clinical trials: the TARGET study.
Murtaza, M., Dawson, S.J., Tsui, D.W., Gale, D., Forshew, T., Piskorz, A.M.,
Nat. Med. 25, 738–743.
Parkinson, C., Chin, S.F., Kingsbury, Z., Wong, A.S., et al. (2013). Non-invasive
analysis of acquired resistance to cancer therapy by sequencing of plasma
Schadendorf, D., van Akkooi, A.C.J., Berking, C., Griewank, K.G., Gutzmer, R.,
DNA. Nature 497, 108–112.
Hauschild, A., Stang, A., Roesch, A., and Ugurel, S. (2018). Melanoma. Lancet
392, 971–984.
Newman, A.M., Bratman, S.V., To, J., Wynne, J.F., Eclov, N.C., Modlin, L.A.,
Liu, C.L., Neal, J.W., Wakelee, H.A., Merritt, R.E., et al. (2014). An ultrasensitive
Scher, H.I., Graf, R.P., Schreiber, N.A., Jayaram, A., Winquist, E., McLaughlin,
method for quantitating circulating tumor DNA with broad patient coverage.
B., Lu, D., Fleisher, M., Orr, S., Lowes, L., et al. (2018). Assessment of the val-
Nat. Med. 20, 548–554.
idity of nuclear-localized androgen receptor splice variant 7 in circulating tu-
Newman, A.M., Lovejoy, A.F., Klass, D.M., Kurtz, D.M., Chabon, J.J., Scherer, mor cells as a predictive biomarker for castration-resistant prostate cancer.
F., Stehr, H., Liu, C.L., Bratman, S.V., Say, C., et al. (2016). Integrated digital JAMA Oncol. 4, 1179–1186.
error suppression for improved detection of circulating tumor DNA. Nat. Bio-
technol. 34, 547–555. Shapiro, B., Chakrabarty, M., Cohn, E.M., and Leon, S.A. (1983). Determina-
tion of circulating DNA levels in patients with benign or malignant gastrointes-
Odegaard, J.I., Vincent, J.J., Mortimer, S., Vowles, J.V., Ulrich, B.C., Banks, tinal disease. Cancer 51, 2116–2120.
K.C., Fairclough, S.R., Zill, O.A., Sikora, M., Mokhtari, R., et al. (2018). Valida-
tion of a plasma-based comprehensive cancer genotyping assay utilizing Shen, S.Y., Singhania, R., Fehringer, G., Chakravarthy, A., Roehrl, M.H.A.,
orthogonal tissue- and plasma-based methodologies. Clin. Cancer Res. 24, Chadwick, D., Zuzarte, P.C., Borgida, A., Wang, T.T., Li, T., et al. (2018). Sen-
3539–3549. sitive tumour detection and classification using plasma cell-free DNA methyl-
omes. Nature 563, 579–583.
Oxnard, G.R., Thress, K.S., Alden, R.S., Lawrance, R., Paweletz, C.P., Cantar-
€nne, P.A. (2016). Association be-
ini, M., Yang, J.C.-H., Barrett, C.J., and Ja Simpson, K.L., Stoney, R., Frese, K.K., Simms, N., Rowe, W., Pearce, S., Hum-
tween plasma genotyping and outcomes of treatment with osimertinib phrey, S., Booth, L., Morgan, D., Dynowski, M., et al. (2020). A biobank of small
(AZD9291) in advanced non-small-cell lung cancer. J. Clin. Oncol. 34, cell lung cancer CDX models elucidates inter- and intra-tumoural phenotypic
3375–3378. heterogeneity. Nat. Cancer. https://doi.org/10.1038/s43018-020-0046-2.

Pantel, K., and Alix- Panabieres, C. (2010). Circulating tumour cells in cancer Siravegna, G., Mussolin, B., Buscarino, M., Corti, G., Cassingena, A., Crisafulli,
patients: challenges and perspectives. Trends Mol. Med. 16, 398–406. G., Ponzetti, A., Cremolini, C., Amatu, A., Lauricella, C., et al. (2015). Clonal
evolution and resistance to EGFR blockade in the blood of colorectal cancer
Pantel, K., and Alix- Panabieres, C. (2019). Liquid biopsy and minimal residual patients. Nat. Med. 21, 827.
disease — latest advances and implications for cure. Nat. Rev. 16, 409–424.
Snyder, A., Morrissey, M.P., and Hellmann, M.D. (2019). Use of circulating tu-
Pantel, K., Hille, C., and Scher, H.I. (2019). Circulating tumor cells in prostate mor DNA for cancer immunotherapy. Clin. Cancer Res. 25, 6909–6915.
cancer: from discovery to clinical utility. Clin. Chem. 65, 87–99.
Soler, A., Cayrefourcq, L., Mazard, T., Babayan, A., Lamy, P.J., Assou, S., As-
Pao, W., and Chmielecki, J. (2010). Rational, biologically based treatment of senat, E., Pantel, K., and Alix-Panabières, C. (2018). Autologous cell lines from
EGFR-mutant non-small-cell lung cancer. Nat. Rev. Cancer 10, 760–774. circulating colon cancer cells captured from sequential liquid biopsies as
model to study therapy driven tumor changes. Sci. Rep. 8, 15931.
Paoletti, C., Cani, A.K., Larios, J.M., Hovelson, D.H., Aung, K., Darga, E.P.,
Cannell, E.M., Baratta, P.J., Liu, C.J., Chu, D., et al. (2018a). Comprehensive Sparano, J., O’Neill, A., Alpaugh, K., Wolff, A.C., Northfelt, D.W., Dang, C.T.,
mutation and copy number profiling in archived circulating breast cancer tu- Sledge, G.W., and Miller, K.D. (2018). Association of circulating tumor cells

494 Cancer Cell 37, April 13, 2020

Cancer Cell

Review
with late recurrence of estrogen receptor–positive breast cancer. JAMA Oncol. Vogelzang, N.J., Fizazi, K., Burke, J.M., De Wit, R., Bellmunt, J., Hutson, T.E.,
4, 1700–1706. Crane, E., Berry, W.R., Doner, K., Hainsworth, J.D., et al. (2017). Circulating tu-
mor cells in a phase 3 study of docetaxel and prednisone with or without lena-
Steensma, D.P., Bejar, R., Jaiswal, S., Lindsley, R.C., Sekeres, M.A., Hasser- lidomide in metastatic castration-resistant prostate cancer. Eur. Urol. 71,
jian, R.P., and Ebert, B.L. (2015). Clonal hematopoiesis of indeterminate po- 168–171.
tential and its distinction from myelodysplastic syndromes. Blood 126, 9–16.
Wang, Z., Duan, J., Cai, S., Han, M., Dong, H., Zhao, J., Zhu, B., Wang, S.,
Stroun, M., Lyautey, J., Lederrey, C., Olson-Sand, A., and Anker, P. (2001). Zhuo, M., Sun, J., et al. (2019). Assessment of blood tumor mutational burden
About the possible origin and mechanism of circulating DNA apoptosis and as a potential biomarker for immunotherapy in patients with non-small cell lung
active DNA release. Clin. Chim. Acta 313, 139–142. cancer with use of a next-generation sequencing cancer gene panel. JAMA
Oncol. 5, 696–702.
Tan, E.M., and Kunkel, H.G. (1966). Characteristics of a soluble nuclear antigen
precipitating with sera of patients with systemic lupus erythematosus. Werner, S.L., Graf, R.P., Landers, M., Valenta, D.T., Schroeder, M., Greene,
J. Immunol. 96, 464–471. S.B., Bales, N., Dittamore, R., and Marrinucci, D. (2015). Analytical validation
and capabilities of the epic CTC platform: enrichment-free circulating tumour
Tannock, I.F., and Hickman, J.A. (2019). Molecular screening to select therapy cell detection and characterization. J. Circ. Biomark. 5, 3.
for advanced cancer? Ann. Oncol. 30, 661–663.
Willis, J., Lefterova, M.I., Artyomenko, A., Kasi, P.M., Nakamura, Y., Mody, K.,
Tayoun, T., Faugeroux, V., Oulhen, M., Aberlenc, A., Pawlikowska, P., and Far-
Catenacci, D.V.T., Fakih, M., Barbacioru, C., Zhao, J., et al. (2019). Validation
ace, F. (2019). CTC-derived models: a window into the seeding capacity of
of microsatellite instability detection using a comprehensive plasma-based
circulating tumor cells (CTCs). Cells 8, https://doi.org/10.3390/cells8101145.
genotyping panel. Clin. Cancer Res. 25, 7035–7045.
Thierry, A.R., El Messaoudi, S., Gahan, P.B., Anker, P., and Stroun, M. (2016).
Wong, F.C., and Lo, Y.M. (2016). Prenatal diagnosis innovation: genome
Origins, structures, and functions of circulating DNA in oncology. Cancer
sequencing of maternal plasma. Annu. Rev. Med. 67, 419–432.
Metastasis Rev. 35, 347–376.

Thress, K.S., Brant, R., Carr, T.H., Dearden, S., Jenkins, S., Brown, H., Ham- Wu, Y.-L., Leeb, V., Liam, C.-K., Lue, S., Park, K., Srimuninnimit, V., Wangh, J.,
mett, T., Cantarini, M., and Barrett, J.C. (2015). EGFR mutation detection in Zhoui, C., Appius, A., Buttonk, P., et al. (2018). Clinical utility of a blood-based
ctDNA from NSCLC patient plasma: a cross-platform comparison of leading EGFRmutation test in patients receiving first-line erlotinib therapy in the
technologies to support the clinical development of AZD9291. Lung Cancer ENSURE, FASTACT-2, and ASPIRATION studies. Lung Cancer 126, 1–8.
90, 509–515.
Wu, T.D., Madireddi, S., de Almeida, P.E., Banchereau, R., Chen, Y.J., Chitre,
€ckstock, J., Andergassen, U., de Gre-
Trapp, E., Janni, W., Schindlbeck, C., Ju A.S., Chiang, E.Y., Iftikhar, H., O’Gorman, W.E., Au-Yeung, A., et al. (2020). Pe-
gorio, A., Alunni-Fabbroni, M., Tzschaschel, M., Polasik, A., Koch, J.G., et al.; ripheral T cell expansion predicts tumour infiltration and clinical response. Na-
SUCCESS Study Group (2019). Presence of circulating tumor cells in high-risk ture. https://doi.org/10.1038/s41586-020-2056-8.
early breast cancer during follow-up and prognosis. J. Natl. Cancer Inst. 111,
380–387. Yu, H.A., Arcila, M.E., Rekhtman, N., Sima, C.S., Zakowski, M.F., Pao, W., Kris,
M.G., Miller, V.A., Ladanyi, M., and Riely, G.J. (2013). Analysis of tumor spec-
Ulz, P., Thallinger, G.G., Auer, M., Graf, R., Kashofer, K., Jahn, S.W., Abete, L., imens at the time of acquired resistance to EGFR-TKI therapy in 155 patients
Pristauz, G., Petru, E., Geigl, J.B., et al. (2016). Inferring expressed genes by with EGFR-mutant lung cancers. Clin. Cancer Res. 19, 2240–2247.
whole-genome sequencing of plasma DNA. Nat. Genet. 48, 1273–1278.
Yu, M., Bardia, A., Aceto, N., Bersani, F., Madden, M.W., Donaldson, M.C.,
Ulz, P., Perakis, S., Zhou, Q., Moser, T., Belic, J., Lazzeri, I., Wölfler, A., Ze- Desai, R., Zhu, H., Comaills, V., Zheng, Z., et al. (2014). Ex vivo culture of circu-
bisch, A., Gerger, A., Pristauz, G., et al. (2019). Inference of transcription factor lating breast tumor cells for individualized testing of drug susceptibility. Sci-
binding from cell-free DNA enables tumor subtype prediction and early detec- ence 345, 216–220.
tion. Nat. Commun. 10, 4666.
Yue, C., Jiang, Y., Li, P., Wang, Y., Xue, J., Li, N., Li, D., Wang, R., Dang, Y., Hu,
Underhill, H.R., Kitzman, J.O., Hellwig, S., Welker, N.C., Daza, R., Baker, D.N., Z., et al. (2018). Dynamic change of PD-L1 expression on circulating tumor
Gligorich, K.M., Rostomily, R.C., Bronner, M.P., and Shendure, J. (2016). Frag- cells in advanced solid tumor patients undergoing PD-1 blockade therapy. On-
ment length of circulating tumor DNA. PLoS Genet. 12, e1006162. coimmunology 7, e1438111.

Valpione, S., Galvani, E., Tweedy, J., Mundra, P.A., Banyard, A., Middlehurst, Zhang, L., Ridgway, L.D., Wetzel, M.D., Ngo, J., Yin, W., Kumar, D., Goodman,
P., Barry, J., Mills, S., Salih, Z., Weightman, J., et al. (2020). Immune-awak- J.C., Groves, M.D., and Marchetti, D. (2013). The identification and character-
ening revealed by peripheral T cell dynamics after one cycle of immuno- ization of breast cancer CTCs competent for brain metastasis. Sci. Transl.
therapy. Nat. Cancer 1, 210–221. Med. 5, 180ra14.

Cancer Cell 37, April 13, 2020 495