Does the mobilization of circulating tumour cells during cancer therapy cause metastasis? Nature reviews Clinical oncology. nrclinonc_2016.pdf

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Does the mobilization of circulating tumour cells during cancer therapy cause
metastasis?
Article in Nature Reviews Clinical Oncology · August 2016
DOI: 10.1038/nrclinonc.2016.128
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Olga A Martin
University of Wollongong
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Patients with the most common carcinomas, such as
those affecting the lung, prostate, bowel or breast, fre-
quently die from distant metastasis, even after curative-
intent surgery, radiotherapy, or chemoradiotherapy
for apparently locoregionally confined disease. Distant
metastases that become apparent after successful treat-
ment to achieve locoregional control are considered to
derive from micrometastases that were present before
treatment commenced1–3
. In patients for whom attempts
to eliminate locoregionally confined disease have failed,
distant metastasis might also derive from residual local
disease4
. Another unwelcome possibility is that metastasis
can sometimes be caused or promoted by the antitumour
treatment itself. Events surrounding treatment with sur-
gery, radiotherapy, and systemic antitumour therapies can
perturb tumours in complex ways that might influence
either positively or negatively the risk of distant metas-
tasis. Evidence indicates that all three modalities might,
under some
circumstances, mobilize tumour cells into
the bloodstream.
Circulating tumour cells (CTCs) can be detected
in patients with all of the major carcinomas and are
necessary for distant metastasis to occur5
. In general,
although patients with high numbers of CTCs have a
poor prognosis6
, the mobilization of CTCs induced by a
therapeutic or diagnostic intervention is not necessarily
associated with an adverse outcome. Metastasis is some-
times regarded as a linear process, whereby tumour cells
acquire enhanced migratory and invasive capacities, enter
lymphatic and blood vessels, attain both the capacity to
survive the harsh environment of the circulation and the
ability to extravasate and proliferate to form a metastatic
lesion, in a new and potentially unfavourable microenvi-
ronment7
. Luzzi et al.8
reported that most CTCs can sur-
vivetheshearforcesthattheyencounterinthecirculation,
enabling them to lodge in capillary beds and often extra
vasate into tissues. The most common outcome in such
situations is cell death, but a small proportion of tumour
cells (approximately 0.02%) survive and, after a vari
able
period of latency, expand into clinically detectable lesions.
In a modelling study, Coumans et al.9
assessed data from a
large cohort of patients with breast cancer and estimated
thatthemetastaticrateismuchlowerthanthatreportedin
thestudydiscussed4
:onlyonemetastasisformsfromevery
60 million cells that disseminate from the primary site.
The time to first metastasis varies widely among
different malignancies, owing to their diverse natural
histories. In many patients with apparently localised
1
Division of Radiation
Oncology and Cancer
Imaging, Peter MacCallum
Cancer Centre.
2
Molecular Radiation Biology
Laboratory, Peter MacCallum
Cancer Centre.
3
Metastasis Research
Laboratory, Peter MacCallum
Cancer Centre, 305 Grattan
Street, Melbourne, Victoria
3000, Australia.
4
The Sir Peter MacCallum
Department of Oncology,
University of Melbourne.
5
Department of Obstetrics
and Gynaecology, University
of Melbourne, Grattan street,
Melbourne, Victoria 3000,
Australia.
Correspondence to M.P.M.
Michael.macmanus@
petermac.org
doi:10.1038/nrclinonc.2016.128
Published online 23 Aug 2016
Does the mobilization of circulating
tumour cells during cancer therapy
cause metastasis?
Olga A. Martin1,2,4
, Robin L. Anderson3,4
, Kailash Narayan1,4,5
and Michael P. MacManus1,4
Abstract|Despiteprogressiveimprovementsinthemanagementofpatientswithlocoregionally
confined,advanced-stagesolidtumours,distantmetastasisremainsaverycommon—andusually
fatal—modeoffailureafterattemptedcurativetreatment.Surgeryandradiotherapyarethe
primarycurativemodalitiesforthesepatients,oftencombinedwitheachotherand/orwith
chemotherapy.Distantmetastasisoccurringaftertreatmentcanarisefrompreviouslyundetected
micrometastasesor,alternatively,frompersistentlocoregionaldisease.Anotherpossibilityisthat
treatmentitselfmightsometimescauseorpromotemetastasis.Surgicalinterventionsinpatients
withcancer,includingbiopsies,arecommonlyassociatedwithincreasedconcentrationsof
circulatingtumourcells(CTCs).HighCTCnumbersareassociatedwithanunfavourableprognosis
inmanycancers.RadiotherapyandsystemicantitumourtherapiesmightalsomobilizeCTCs.We
reviewthepreclinicalandclinicaldataconcerningcancertreatments,CTCmobilizationandother
factorsthatmightpromotemetastasis.Contemporarytreatmentregimensrepresentthebest
availablecurativeoptionsforpatientswhomightotherwisediefromlocallyconfined,
advanced-stagecancers;however,ifsuchtreatmentscanpromotemetastasis,thisprocessmust
beunderstoodandaddressedtherapeuticallytoimprovepatientsurvival.
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cancers, metastasis occurs early, even before diagnosis3
.
This discovery has invoked an alternative parallel pro-
gression model of metastasis, whereby tumour cells that
disseminate early evolve independently, but in parallel,
from the primary tumour10,11
. Disseminated tumour
cells might lie dormant in bone marrow and/or other
tissues for many years. These occult cells can enter a
G0-like state and remain as quiescent single cells, or
they can expand to form a cluster that is unable to
expand further, possibly owing to effective immune
surveillance, or the inability to trigger angiogenesis12
.
In either case, these micrometastases are presumed to be
the source of the CTCs that are detected after primary
tumour control in many patients. Genomic analyses13,14
have reported ‘early’ and ‘late’ metastases, an observa-
tion confounded by the fact that a metastasis can seed
secondary metastases.
Curing the most-common epithelial and mesen-
chymal malignancies is generally impossible without
loco
regional control provided by surgery and/or radio-
therapy; however, if such potentially curative therapies
are also able to mobilize tumour cells into the circulation,
or to promote the growth of dormant micro
metastases,
a detailed understanding of these events is essential.
This knowledge could lead to the design of new thera
peutic approaches capable of increasing the cure rates
for
cancers for which metastasis is a frequent cause of
treatment failure. In this Review, we examine the pre-
clinical and clinical effects of the major modalities used
in curative treatment of epithelial cancers on the mobil
ization of tumour cells into the circulation, and consider
other treatment-related factors that could potentially
modulate the risk of metastasis. When evaluating these
data, we have considered in vitro and animal studies sep-
arately from clinical studies, with the latter constituting a
higher level of evidence because of their direct relevance
to patients.
Circulating tumour cells
Both CTCs and cell-free tumour-derived DNA are com-
monly found in the blood of patients with carcinoma,
including those who will never develop overt metastatic
disease. This observation reflects the extremely inefficient
natureofmetastasis8
.Despitethisinefficiency,thenumber
of CTCs and cell-free tumour DNA found in the circula-
tion has both prognostic and predictive value in several
different cancer types15–18
.
CTCs escape from the primary tumour environment
through an active process, in which they acquire expres-
sion of genes involved in migration and invasion, and of
genes that switch their morphology to a mesenchymal-
like motile phenotype. Epithelial-to‑mesenchymal tran-
sition (EMT) has been investigated in many settings and
evidence for its role in driving the release of CTCs is
growing19,20
. However, EMT is not essential for tumour
cell escape; an elevated intratumour interstitial pressure,
an incompetent or damaged tumour vasculature, and
migration of actively motile cells can collectively result in
the entry of single CTCs or clusters of cells into the termi-
nal lymphatics or bloodstream21–23
. Both in clinical sam-
ples and in preclinical tumour models, tumour cells have
been shown to be associated with macrophages during
migration towards blood vessels, where they interact with
endothelial cells before intravasation24,25
. The presence of
CTCs with both epithelial and mesenchymal properties
has been reported in blood samples from patients with
breast20,26
, prostate26
and lung27

cancer; cell clusters, com-
posed in part of tumour cells, have also been detected
in the circulation of these patients, and their presence
is associated with an earlier onset of metastatic disease

compared with single CTCs28–31
.
The ability of CTCs to lodge in capillaries and expand
to form macrometastases has been demonstrated in sev-
eral studies, including a report using tumour xenografts
generated from blood samples of patients with metastatic
prostate or colon cancer32
. In subsequent studies, CTCs
from patients with breast and small-cell lung
cancer
(SCLC) injected into immunologically
compromised
mice resulted in tumour formation33–35
.
Researchers have reported that breast and prostate
CTCs can be propagated in vitro for prolonged
periods
of time (>6 months in some cases) as spheroids or
organoids32,36
— a hallmark of cancer stem cells (CSCs)
that are known to have a high capacity for self-renewal,
a radioresistance and chemoresistance phenotype, and a
high metastatic potential37,38
. The cell lines and tumours
derived from these CTCs share histological and immuno
chemical features and a mutational landscape similar
to the primary tumour, indicating that their biologi-
cal properties remain after long-term culture. In one
study35
, multiple tumour cell lines could be established
from six out of 36 patients with breast cancer from whom
CTCs had been obtained during chemotherapy, but not
from CTCs harvested before treatment, indicating that
the CTCs isolated during treatment were more numer-
ous and/or more autonomous in their growth require-
ments than those obtained before treatment. Taken
together, these data demonstrate that, in addition to
being prognostic or predictive markers, CTCs can also
be metastasis-initiating cells.
While, theoretically, CTCs can access all tissues in the
body, secondary tumours tend to arise in selected tissues,
depending on the type of cancer. This observation is the
basis of the famous ‘seed and soil’ hypothesis proposed
over 125 years ago by Stephen Paget39
. Studies conducted
Key points
• Distant metastasis remains a frequent cause of death, even after locoregional
disease control is achieved using surgery, radiotherapy, and/or systemic therapy
• Antitumour therapies can, under some circumstances, mobilize tumour cells into the
peripheral circulation that might influence the risk of distant metastasis
• Irradiation can enable tumour cells to acquire properties that facilitate their
dissemination and the subsequent generation of metastases
• Severalmechanismsmightexplainametastasis-promotingeffectderivedfromsurgical
procedures,suchastissuedisruptionandleakageofbloodcontainingtumourcells
• A potential link between systemic therapies and metastasis has not been established,
but the results of isolated studies indicate that this question needs to be addressed
• The risk of distant failure from antitumour therapy can potentially be reduced if
treatment-related factors capable of promoting metastasis are identified, and
targeted therapeutically
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over the past decade have indicated that CTCs ‘home’
to sites within tissues in which tumour-promoting stro-
mal cells — including cells of the myeloid, fibroblast and
endothelial lineages — have already been recruited
and provide a supportive niche for the tumour cell40,41
.
The proposed mechanism by which the primary tumour
initiates the formation of the premetastatic niche is by
secreting factors, either as freely circulating molecules or
encapsulated within exosomes, that direct stromal cells to
tissue sites (or modulate the phenotypes of cells resident
at the site) that later attract CTCs42,43
. Whether the for-
mation of a niche is essential for metastases to develop,
and whether the early arrival of CTCs might stimulate the
initiation of the niche remains to be determined.
Radiotherapy
Preclinical studies
In the 1940s, the suspicion that an increased rate of
metastasis might follow radiotherapy led to testing of this
hypothesis in animal tumour models. The initial investi
gations, summarized by von Essen44
, involved a range
of tumour-bearing animals irradiated at vari
ous doses
in single or multiple fractions. In general, these
studies
indicated that irradiation of primary tumours with lower
doses (insufficient for local
control) was associated
with the highest risk of metastases. Kaplan and Murphy45
observed an unexpectedly high number of early distant
metastases in patients with epidermoid carcinoma of the
lower lip and buccal mucosa treated with radiotherapy.
In 1949, they conducted a series of animal experiments
using subcutaneously transplanted murine mammary
carcinomas that were locally irradiated with 4–10Gy
(non-curative) in single X‑ray doses. Within 8 weeks,
pulmonary metastases were found in 43.5% of irradiated
mice and 9.6% of controls. Those metastases were exclu-
sively pulmonary and grew intravascularly in arteries
and arterioles. The irradiated tumours recurred locally
within 2 weeks. In 1976, Sheldon and Fowler46
irradiated
mammary carcinomas implanted on the anterior chest
wall of syngeneic mice using either a single dose of 5Gy
or two fractions of 3.5Gy; excision of the tumours was
performed while the mice were under anaesthesia for the
second (real or sham) irradiation. Pulmonary metastasis
occurred 10 weeks later in 20% of non-
irradiated mice,
25% of mice that received 5Gy and 30% of those that
received two doses of 3.5Gy, indicating that the metas-
tasis rate was enhanced by
two-fraction irradiation

compared with surgical removal alone.
In the past 15 years47–51
, investigators have sought to
explain this phenomenon. For example, Camphausen
and colleagues47
injected Lewis lung carcinoma cells
into the hindlimbs of mice and irradiated the resulting
tumours. Local disease control was achieved in 71%
of the irradiated mice, but these mice had a higher
number of lung metastases when compared with non-
irradiated mice. In further experiments involving mice
that had been injected with fibrosarcoma-derived cells,
irradiation markedly increased the numbers of pul-
monary metastases compared with those detected in
non-
irradiated tumour-bearing mice. The researchers
suggested that previously dormant metastases had been
activated by radiation47
, in agreement with von Essen
and colleagues44
, who showed that both tumour
cells and stroma are modified by radiation, promoting
the selection of
more-aggressive and invasive cells with
metastatic potential.
Studies of tumour cell biology indicate that radio
therapy can alter tumour cells, making them more
aggressive than non-irradiated cells. The two phases of
a typical course of conventionally fractionated high-dose
radiotherapy (~2Gy per fraction) can have different bio-
logical effects (FIG. 1). In the later phase of radiotherapy
(for example, after 30 daily fractions of 2Gy), the high
cumulative radiation doses will have caused permanent
loss of reproductive capacity in all clonogenic tumour
cells, if the treatment is successful. Tumour cell death
occurs primarily through the induction of irreparable
DNA damage and cell cycle arrest52
. Irradiated cells usu-
ally undergo one or two error-prone cell cycles before
succumbing to mitotic catastrophe, apoptosis, necrosis, or
autophagy53–55
, depending on the dose. In the early stages
of fractionated radiotherapy (that is, after delivery of only
sublethal cumulative radiation doses, such as 2–6 Gy in
1–3 fractions), a significant proportion of the irradiated
tumour cells can repair the DNA damage; tumour cells
are much more likely to survive if they escape into the
circulation at this stage than the more-heavily irradiated
cells that enter the circulation later in treatment.
Cellular radioresistance and radiosensitivity depend
on many factors, including cell type and origin, cell-cycle
phase, tissue oxygenation, and genetic background. CSCs
are more radioresistant than non-stem cells56,57
and can
selectively survive irradiation. If they are not elimi
nated
or do not undergo permanent cell-cycle arrest (senes-
cence or dormancy), CSCs have the capacity to cause
disease recurrence. Furthermore, non-small-cell lung
carcinoma (NSCLC)-derived cells have been reported to
survive 5Gy irradiation by acquiring new CSC charac
teristics, including the capacity to form 3D spheroids
in vitro, to self-renew and to generate differentiated
progeny. These cells also acquire the more-mesenchymal
phenotype typical of EMT38
.
Failure to repair DNA double-strand breaks caused
by radiotherapy is a major threat to genomic integrity58,59
.
Irradiated tumour cells have increased genome instability
and plasticity compared with unirradiated cells, and thus
can become more radioresistant. Rofstad et al.48
reported
that irradiation of mouse melanoma xenografts induced
tumour hypoxia that, in turn, promoted metastasis by
upregulating the expression of urokinase plasminogen
activator surface receptor (uPAR). Injections of anti-
uPAR antibodies almost completely blocked metastasis.
Changes in invasiveness and biomechanical properties
might also occur in irradiated cells. For example, the
expression levels of the proto-oncogene MET (encoding
HGFR, a known driver cell invasiveness), were fivefold
higher in tumour cells irradiated at 10Gy than in non-
irradiated tumour cells60
; silencing of MET expression
prevented radiation-induced proliferation and invasive-
ness,andpromotedapoptosis.Zheng et al.61
,reportedthat
low-doseirradiation(1–4Gy)ofcellsderivedfromtongue
squamous-cellcarcinomaincreasedtheirinvasivenessina
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dose-dependent manner and changed some of their bio-
mechanical properties, including the organization of their
actin cytoskeleton.
In 2015, a study revealed that the migratory capacity of
tumour cells induced by low doses of radiation was medi-
ated by G‑CSF secreted by the tumour cells62
. Human
NSCLC cells expressing G‑CSF receptors were stimulated
with radiation to release G‑CSF, which subsequently pro-
moted EMT through an autocrine mechanism involving
JAK/STAT3 signalling62
. Radiotherapy can also stimu-
late primary tumour self-seeding from CTCs released
either before or after radiation exposure. Vilalta and
co-workers63
used preclinical models to demonstrate the
release of GM‑CSF from irradiated tumour cells stimu
lated the migration of tumour cells into the irradiated site,
thus triggering local tumour recurrence.
Abundant evidence indicates that radiation can indi-
rectly affect metastasis by modulating angio
genesis,
thereby affecting the biological behaviour of tumour
cells. For example, radiation stimulates hypoxia-inducible
factor 1 (HIF‑1) and, subsequently, the expression
of VEGF64,65
. In a clinical trial in which patients with
hepato
cellular carcinoma received either chemotherapy
alone or chemotherapy plus conformal radiotherapy, an
associationwasobservedbetweencombinedchemoradio
therapy and rapid tumour progression outside the irradi
ated field66
. In cell-culture experiments performed in
hypoxia-mimicking conditions, Sofia Vala et al.67
demon-
strated that low doses of radiation (<0.8 Gy) led to the
rapid phosphorylation of VEGFR‑2 and induced VEGF
production — thereby, enhancing endothelial-cell migra-
tion. Moreover, Shen et al.68
reported that irradiation of
cultured cancer cells induces secretion of protein-lysine
6‑oxidase (LOX), a key player in hypoxia-dependent
tumour-celldisseminationandmetastasis.IncreasedLOX
secretion was also detected in mouse serum after tumour
xenografts were exposed to localized radiation63
.
Clinical studies of radiotherapy
In some clinical situations, locoregional radiotherapy
can reduce the risk of distant metastasis. For example, in
patients with stage I–III breast cancer, adjuvant regional
radiotherapy to the draining lymph nodes is associated
with a substantial improvement in distant metastasis-
free survival (hazard ratio (HR) 0.82), leading to a sig-
nificant improvement of overall survival (HR 0.85, 95%
CI 0.75–0.96; P=0.011) at 10 years69
. Nevertheless, our
group reported the first clinical evidence that localized
radiotherapy, when delivered to an intact tumour, can
mobilize viable tumour cells into the circulation31,70
,
a phenomenon that could potentially increase the risk
of distant metastasis. We detected increased numbers of
CTCs, both as single entities or in clusters, in the blood
of patients with NSCLC early in the course of their radio
therapy. Intense staining for phosphorylated histone
H2AX (γ-H2AX), a biomarker for DNA double-strand
breaks71,72
, showed that mobilized CTCs were derived
from the irradiated tumour, with the highest number of
CTCs mobilized after the first and second daily fractions
of radiotherapy — a sublethal dose for many tumour
cells (FIG. 1). Mobilized CTCs had an increased capacity
to grow in culture both as attached cultures and orga-
noids, compared with CTCs collected before treatment,
(O. A. Martin, unpublished work); such attributes can be
associated with unfavourable patient outcomes34
.
In lung cancer, strong clinical evidence indicates
a link between the fractionation schedule employed
in radiotherapy and the subsequent risk of distant
Nature Reviews | Clinical Oncology
Radiation-induced
DNA damage
Cancer
non-stem
cells
Cancer
stem cells
DNA damage repaired
DNA damage unrepaired Radiation-induced
cell death
DNA damage misrepaired
• EMT
• Hypoxia
• Motility
• Invasiveness
Initial 2 Gy
radiotherapy
fractions
Accumulated radiotherapy fractions
Increasing number of treatment fractions over time
Early radiotherapy
survival (<50%)
Figure 1 | Progressive effects of fractionated radiotherapy on tumour cells in vivo. Up to one-half of the malignant
cells in an irradiated tumour can survive the first radiotherapy fractions; they can subsequently acquire a more-aggressive
phenotype, becoming circulating tumour cells that are detectable during the course of radiotherapy. Radiotherapy affects
the regulation of genes associated with radioresistance, tumour aggressiveness, and enhanced metastatic potential,
including signatures associated with hypoxia, invasiveness and motility, and epithelial-to‑mesenchymal transition (EMT).
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metastasis. Continuous hyperfractionated accelerated
radiotherapy (CHART)73
, a very intense but brief treat-
ment regimen, improved survival and local disease con-
trol compared with conventional prolonged radiotherapy
in patients with locally advanced NSCLC. CHART was
also associ
ated with a 24% reduction in the relative risk
of death, equivalent to an absolute improvement in 2‑year
survival from 20% to 29% (P=0.004) between conven-
tional radiotherapy and CHART. With mature follow‑up
(at least 3 years), in patients with squamous carcinoma
a 24% reduction in the relative risk of distant metasta-
sis was noted (P=0.043)74
. The reduction in the distant
metastatic rate in patients treated with CHART has sev-
eral explanations, including a reduced probability of late
metastasis from uncontrolled, compared with controlled,
local disease. CHART was delivered in three fractions per
day; CTCs mobilized by this radiotherapy regimen pre-
sumably acquired more damage at any given time than
cells exposed to daily radiotherapy — and might have a
reduced capacity to form metastases.
To our knowledge, no other research group has sys-
tematically explored changes in CTCs during the early
phase of radiotherapy. Using a telomerase-based assay,
Dorsey et al.75
detected viable CTCs in serial blood
samples of patients with NCSLC receiving radiotherapy
(telo
merase contributes to cancer cell immortality and is
present in almost all cancer cells, but at very low levels
in normal cells). Lowes et al.76
investigated CTCs using
the CellSearch system (Janssen Diagnostics, Raritan, New
Jersey, USA) in patients with prostate cancer before and
3 months after completion, but not during radiotherapy;
patients who had increased or unchanged CTC numbers
after radiotherapy experienced treatment failure. These
preliminary data76
are derived from a combined popu
lation of only 19 patients, but are consistent with the
hypothesis that failure to clear CTCs might be associated
with an unfavourable prognosis, an important subject for
future research.
Effects of radiotherapy on immunity
Immune responses can be finely balanced between pro-
moting metastasis and inhibiting signalling pathways
affected in cancer; antitumour therapy can perturb this
balance, with a positive or negative effect on the risk
of metastasis77
. Systemic immunosuppression owing
to antitumour therapy, including radiotherapy, could
potentially increase the risk of metastasis. Individuals
who have received renal and cardiac transplants have an
increased incidence of cancer compared with the gen-
eral population, owing to immunosuppressive thera
pies associated with transplantation. In a study with
381 patients who received a cardiac transplant, 130
(34%) had developed a malignancy after a median fol-
low up of 9.7 years78
. Immunosuppressed patients have
a worse prognosis than immunocompetent patients
with cancer79
. Radiotherapy induces a pronounced
local inflammatory response77
, enhancing expression of
inflammation-related cytokines, including TGFβ, which
mediates EMT in mammalian cells80
. Several studies
have examined radiation-induced EMT. For example,
Park et al.49
established a mouse xenograft model using
a cancer cell line expressing luciferase. After irradiation
of primary tumours, the authors observed luminescent
signals identified as intestinal and pulmonary metas-
tases; these lesions expressed EMT markers and were
histologically confirmed as metastatic tumours. The
expression of several proteins50,81–83
has been associated
with the acquisition of EMT characteristics by irradiated
tumour cells, and could be potentially targeted to atten-
uate the harmful effects of radiation and/or to enhance
the efficacy of radiotherapy.
Traditionally, ionizing radiation has been considered
immunosuppressive because of the inherent susceptibil-
ity of naive immune cells to radiation84
and thus, radio
therapy was not deemed to contribute to anti
tumour
immunity. A growing body of evidence, however, sup-
ports the possibility of irradiated tumour cells becom-
ing a robust source of neoantigens, which can prompt
local and systemic antitumour immune responses, in
addition to direct radiation-induced lethality77
. Immune
responses can depend greatly on the type of radiation-
induced cell death (that is, mitotic catastrophe, apoptosis,
necrosis, or autophagy). DNA released from irradiated
tumour cells activates both innate and adoptive immune
cells via different mechanisms85–87
, including boosting
cytotoxic T lymphocytes that contribute to tumour-cell
eradication77
. The fate of a cell damaged by radiation
depends mainly on the extent and type of DNA damage
and, therefore, on the absorbed dose of radiation and the
regimen used88
. The DNA-damage response and signal-
ling pathways involved in the immune response have
been proposed to cooperate to cause cancer cell death89
.
While lower radiotherapy doses (<2 Gy) can promote
anti-inflammatory responses77,90
, higher doses (>15 Gy)
might induce an intense inflammatory response that can
influence the survival of residual tumour cells inside and
outside of the irradiated area91
. Thus, a carefully selected
combination of radiotherapy fractionation schedule and
immune-modulating agents could enhance systemic
antitumour immune responses.
The most intense form of large-fraction radiotherapy
is stereotactic ablative body radiotherapy (SABR),
which is leading to a revolution in the management
of oligometa
static solid tumours and small primary
tumours unsuitable for surgical resection, especially in
lung cancer92,93
. SABR can provide non-inferior survival
outcomes and local disease control comparable to sur-
gery94,95
owing to improvements in linear-accelerator
technology, cancer imaging, and radiotherapy-
planning
software. The delivery of very large radiotherapy frac-
tions (>20 Gy), leads to high levels of cell death in both
tumour and stromal cells — thereby justifying the title
‘ablative’. The profound local inflammatory response
resulting from rapid cell death by SABR likely contributes
to both local control and induction of a systemic immune
response. This response can sometimes be capable of
causing disease regression at sites distant from the irradi
ated volume, the so‑called ‘abscopal’ effect of radiother-
apy96
. The potential effect of SABR on CTC mobilization
has not been studied; future comparisons of the rates of
distant metastasis in matched patients treated with either
surgery or SABR will be of particular interest.
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Radiotherapy–metastasis interactions
Direct evidence for mobilization of CTCs during radio
therapy in humans is at present limited to reports from
our own group in patients with NSCLC31
. This mobil
ization might be due to the effect of radiation on tumour
architecture,especiallyonthetumourvasculature.During
fractionated radiotherapy, actively proliferating tumour
and stromal cells (both radiosensitive) increasingly
undergo cell death with accompanying disruption of
tumour architecture, which could promote direct entry
of tumour fragments into the circulation. Furthermore,
tumour debris, including irradiated stromal and viable
neoplastic cells, might be transported to the terminal lym-
phaticsandsubsequentlyformcoloniesindraininglymph
nodes, owing to mutations or other adaptations enabling
them to survive in this harsh environment. Viable tumour
cells might also be capable of forming distant metastases
upon entry into the
peripheral circulation (FIG. 2).
Patients with detectable lymph-node metastasis at
the time of locoregional treatments are much more likely
to experience distant failure than patients with node-
negative cancer. For example, in a multivariate analysis
of known prognostic factors, only lymph-node involve-
ment was associated with distant relapse in patients with
locally advanced cervical cancer treated with definitive
radiotherapy97
. Similarly, the distant site relapse rate in
patients with endometrial cancer following curative treat-
ment was >66% when both lymphovascular invasion and
lymph-node metastasis were present, but only 7% when
these parameters were negative98
.
Radiotherapy might also negatively affect CTC biol-
ogy. In NSCLC, radiotherapy increased the number of
CTCs detected in clusters31
, which are associated with
an increased rate of metastasis in several cancers30,99,100
.
Radiotherapy was also associated with an increase in
the number of CTCs displaying mesenchymal-like
characteristics31
.
No direct clinical evidence exists to link an increase in
radiotherapy-released CTCs or mesenchymal-like CTC
characteristics associated with a worse prognosis. If the
Primary
lymph node
Secondary
lymph node
Direct tumour
cell entry into
draining veins
Venus circulation
via LV anastomoses
Venus circulation
via LV anastomoses
Venus circulation
via thoracic duct
Disrupted tumour
blood vessel
• Disrupted tumour architecture
• Elevated tumour interstitial pressure
• Opening of lymphatics
• Entry of tumour components into lymphvasculature
a Venous
b Lymphatic
Figure 2 | Routes of cancer cell mobilisation during treatment of an intact tumour. Tumour irradiation with doses
insufficient to eliminate all tumour cells can result in the disruption of the tumour architecture and, subsequently, lead to
the entry of tumour cells and other tumour components into the peripheral circulation, either directly into the venous
system (panel a) or indirectly via the lymphatic system (panel b) through lymphaticovenous (LV) anastomoses, or the
thoracic duct, following hydrostatic pressure gradients. Other therapeutic interventions can have a similar effect on
tumour cell mobilization.
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release of CTCs during radiotherapy does have prog-
nostic value, then strategies designed to target CTCs or
to ensure that they are critically damaged before entry
into the circulation would become appealing. Such strat
egies could include delivery of radiotherapy with larger
or more-frequent fractions, appropriately timed co‑
administration of systemic agents to eliminate mobilized
CTCs,immunemodulationtoeliminateCTCs,andthera
pies targeted at the prevention of EMT or
elimination of

mesenchymal-like tumour cells.
Surgical procedures
Patients with cancer can undergo a wide variety of pre-
surgical and surgical procedures — ranging from external
manipulation (such as endorectal ultrasound) to radical
resection with extensive lymph-node dissection — all of
which have the potential to perturb the tumour and lead
to the release of CTCs. After resection, the surgical bed
can contain, in addition to viable tumour cells, a mixture
of blood, extracellular fluid and a wide range of cytokines
and inflammatory cells, which can promote entry of via-
ble tumour cells into the local and regional lymphatic
vessels and peripheral circulation101
. Tumour cells can be
inadvert
ently implanted into surgical wounds and drain-
age tube tracks, leading to loco
regional recurrence102
.
Several groups have reported that increased numbers of
CTCscanbedetectedduringandaftersurgery(compared
with the levels before surgery), leading to the hypoth
esis that surgery itself could promote distant metastasis.
Invasive surgical procedures are generally performed
under some form of regional or general anaesthesia; the
physiological effects of anaesthetic and surgical pro
cedurescouldpotentiallyinfluencetheriskofmetastasis103
.
Perioperative factors, such as paracrine and neuroendo-
crine responses to surgery, could facilitate the metastatic
process by directly affecting malignant tissues, or through
indirect pathways, such as immunological perturbations.
Surgery-related anxiety and stress, and the nutritional sta-
tus might also be important factors104
. Anaesthesia itself
might also impair various immune functions, including
those of neutrophils, macrophages, dendritic cells, T‑cells,
andnaturalkiller(NK)cells105
.Intravenouslyadministered
induction agents (such as ketamine and thiopentone)
and inhalational agents (such as halothane) can suppress
NK‑cell activity in animal studies, and the upregulation of
hypoxia-inducible factors by volatile anaesthetics can be
cytoprotective for residual tumour cells106
. The pathways
involved in these processes, as well as their clinical rele-
vance,haveyettobeelucidated.Finally,bloodtransfusions
given in the perioperative period have been shown to be
associated with higher disease recurrence (odds ratio 1.6)
in patients with head and neck cancer and hepatocellular
carcinoma,aphenomenonattributedbysomeresearchers
to immunosuppression107
.
Preclinical studies
Detailed reviews on the effect of surgery on tumour
growth and metastasis using preclinical models have
been published108,109
. Evidence supports both a favour-
able and an unfavourable role of surgery in cancer out-
comes. Studies that conclude a detrimental effect of
surgery on subsequent outcome in preclinical models
invoke Folkman’s reports of the release of antiangiogenic
proteins (such as angiostatin and endostatin) from the
primary tumour to prevent the expansion of metastatic
lesions110
. Removal of the primary tumour relieves this
brake on angiogenesis, thus accelerating metastasis.
In other
studies, stimulation of angiogenesis through
wound-healing mechanisms triggered by the surgical
procedure has been demonstrated111
. A study from 2014
that used a preclinical cancer model112
described that the
procedure of core-
needle biopsies can induce distant
metastasis, mediated by inflammatory cytokines secreted
as a result of the wound112
.
Effect of surgery on patients’ CTCs
Biopsies and other diagnostic procedures. Direct evi-
dence has linked needle biopsies (for example, in patients
with prostate cancer3,113,114
), and incisional biopsies (for
example, in patients with oral cancers115,116
), with the
detection of increased numbers of CTCs in the circula-
tion.Zoubekandcolleagues117
reportedachildwithpelvic
Ewing sarcoma who had CTCs detected in the peripheral
blood collected during open tumour biopsy (identified
using RT‑PCR of specific hybrid transcripts), but not
before nor 6 days after surgery117
. Tumours can also form
along the track left by a biopsy needle, as described in a
case report118
. Jones and colleagues119
retrospectively ana-
lysed patients with colorectal cancer liver metastases who
had undergone resection with or without a preoperative
needle biopsy. These investigators reported evidence of
tumours along the needle track in 17% of patients who
had undergone biopsy. The survival rate was worse in
patients who had undergone preopera
tive biopsies com-
pared with those patients who had not119
. A remarkably
congruent local dissemination rate of 16% after liver
biopsy was reported in a similar trial120
. In response to
the increased awareness of tumour seeding along needle
tracks, ‘anti-seeding technology’ using radiofrequency
pulses has been proposed as a method to sterilize the
needle and its track121,122
. In prospective studies, Koch
and colleagues reported that the proportion of patients
having detectable CTCs increased after colonoscopy123
and endorectal ultrasound124
.
Cancer surgery. The number of publications relating
CTCs, surgery, and metastasis risk has increased con-
siderably over the years; herein, we discuss a selection
of those studies. The effects of surgery on CTCs have
been investigated extensively in patients with gastro-
intestinal tumours, especially in those with colorectal
cancer (CRC)125
. CRC cells often disseminate through
draining nodes and vessels; the most frequent distant
meta
static site in patients with CRC is the liver, which
can be accessible by laparotomy. The presence of both
CTCs and disseminated tumour cells in lymph nodes
and bone marrow has negative prognostic significance
in CRC126,127
. Increased numbers of CTCs have been
reported during and after surgery compared with base-
line levels128
. At the time of surgery for CRC, CTCs
can reach the liver through the portal venous circula-
tion; CTCs are detected more frequently and in higher
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numbers in the portal venous compartment than in the
central venous compartment during surgery for CRC129
.
Trapping of CTCs in the liver, however, does not neces
sarily lead to liver metastases. Koch and colleagues130
identified CRC-derived cells in liver biopsies of 10 of 100
patients with UICC stage I–III CRC, but no correlation
between the presence of these cells and the survival rate
of the patients was found, suggesting that most tumour
cells trapped in the liver did not survive to cause metas-
tasis. In a study of 37 patients who underwent resection
of liver metastases131
, however, multi
variate analysis
confirmed that tumour-cell detection in intraoperative
blood and in bone marrow were independent prognos-
tic factors for tumour relapse. Interestingly, Wind et al.131
reported a difference in CTC mobilization between open
and laparo
scopic surgical resections, with fewer CTCs
detected after laparoscopy.
If CTCs mobilized by surgery in CRC are, indeed, a
cause of metastases, then preoperative treatment could
potentially modulate the metastatic risk. The number of
CTCs detectable during and after surgery can be reduced
by the use of preoperative chemoradiotherapy in rectal
cancer132
. Kienle and colleagues132
assessed the blood and
bone marrow samples from 142 patients collected before,
during, and after surgery. Tumour cells were detected
in 34 of 103 (33%) bone marrow and 65 of 117 (55.6%)
blood samples of patients who did not receive treatment,
compared with 4 of 24 (16.7%) bone marrow and 10 of
25 (40%) blood samples of patients receiving treatment.
The tumour-cell detection rate was lower in the chemo-
radiotherapy group compared with untreated patients,
but the survival rate was significantly worse in patients
with tumour cells in the bone marrow after neoadjuvant
therapy compared with those without detectable bone
marrow involvement132
. Preoperative radiotherapy and
chemoradiotherapy both improved local disease control,
but the roles of these treatments in metastasis prevention
or overall-survival improvements is unclear133
. In a study
of 162 patients with rectal cancer treated with preopera-
tive radiotherapy, Nesteruk and colleagues134
found that
thedetectionofCTCs7daysaftersurgerywasofprognos-
tic value for local recurrence, whereas detection of CTCs
before or 24 hours after surgery was not.
In breast cancer, a bimodal pattern of distant metas-
tasis after treatment has been reported in surgical studies
involving large cohorts (ranging between 1,526–3,921
patients)135–137
, a phenomenon that could be related
to events occurring at the time of surgery. Demicheli
and colleagues138
analysed the results of trials in which
patients with axillary node-positive breast cancer had
received either adjuvant chemotherapy after breast

cancer surgery, or mastectomy and nodal dissection
alone. These investigators proposed that the meta-
static process could be driven by mastectomy, and that
micrometastases could undergo sustained periods
of dormancy.
In NSCLC, both open surgery and video-assisted
surgery139,140
have been associated with increased CTC
counts in venous blood. Hashimoto and colleagues141
found that, on average, surgery caused increases of CTC
numbers in the pulmonary veins in 30 patients treated
with lobectomy for peripheral NSCLC. Dong and col-
leagues142
studiedacohortof31patientswithNSCLCwho
had blood collected from a pulmonary vein during open
thoracic surgery; of these 31 patients, 15 had positive test
results for CTCs. The median survival and 2‑year survival
rates for patients with positive versus negative findings
were11monthsand26.7%, respectively,versus27months
and 62.5%, respectively.
In organ-confined prostate cancer, Eschwege et al.143
reported that CTC numbers were increased in 12 of 14
patients who underwent surgery compared with before
surgery. Other situations in which CTC counts might
become elevated during or soon after surgery include
breast cancer resection144
, transurethral bladder cancer
resection145
and oesopha
geal cancer resection146
. The
surgical community has become increasingly aware of
the potential association between surgical intervention,
CTCs and metastasis risk, and is actively seeking ways to

investigate and reduce CTC mobilization.
Surgery–metastasis interaction
The mechanisms by which therapeutic interventions
can promote CTC mobilization are unknown and are
likely to vary between and within modalities. The worst
outcomes observed in patients with mobilized CTCs
might reflect the negative impact of underlying vascular
invasion rather than a sudden large influx of CTCs. For
example, mobilization of CTCs after rectal ultrasound,
which involves external pressure on the tumour, could
reflect an early release of CTCs already primed within
blood vessels. Direct extension of macroscopic tumours
into large veins is an extreme example of such vascular
invasion (FIG. 3). In renal carcinomas, life-
threatening
tumour embolisation to the lung can occur, before
or during surgical resection147
. In patients receiving
curative-
intent surgery, the severing of blood and lym-
phatic vessels in proximity to the tumour can lead to the
formation of a fluid-filled cavity containing tumour cells
that might subsequently gain access to draining lym-
phatic channels148
. In addition, surgery induces a local
inflammatory response in the wound, characterised by
the influx of immune cells and a release of cytokines that
can
influence the risk of metastasis149
.
a b
SVC
SVC
T
T
A
A
Figure 3 | Tumour extension into the venous system. a | Coronal CT and
b | 18
F-FDG-PET/CT images of a patient with direct extension of a lung cancer (T) into
the superior vena cava (SVC). The tumour causes a filling defect within the SVC and,
unlike a simple clot (which would be metabolically inactive), is FDG-avid.
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A causal link between surgery and metastasis has not
beenestablishedtodate.Nevertheless,proposedstrategies
designed to minimise the entry of CTCs into the vascu-
lar system at the time of surgery, include clamping the
blood vessels that drain the tumour at the earliest possible
time point, and isolating the tumour from the circulation
before resection150
.
Systemic antitumour therapies
Evidence from studies of both surgery and radiotherapy
indicates that disturbance of the tumour or its environ-
ment by therapeutic intervention might, under some
circumstances, promote metastasis. Our group has, there-
fore, investigated the possibility that systemic anticancer
therapies might also occasionally have this effect, despite
the counterintuitive nature of such a hypothesis. A very
limited number of publications have addressed this topic.
Unlike surgery and radiotherapy, systemic treatments for
cancer — including chemotherapy and targeted thera-
pies — can have a direct effect not only on cancer cells in
the primary tumour, but also on CTCs and micrometas-
tases present at the time of treatment. In a growing num-
ber of clinical studies, the CTC count has been used to
monitor response to systemic therapy, but the potential
prognostic and predictive value of CTC release early dur-
ing chemotherapy or targeted therapies has not been fully
explored. In many studies, the presence of CTCs before
chemotherapy is associated with a worse overall survival,
compared with that of patients in whom CTCs are not
detectable initially151
. Smerage and colleagues151
reported
that, in patients with increased CTC counts (≥5 CTCs per
7.5ml of whole blood) detected before the commence-
ment of chemotherapy, the number of CTCs often failed
to decrease after treatment. Paradoxically, the results of
somepreclinicalandclinicalstudiesonthemobilizationof
haematopoieticstemcellsbeforehigh-dosechemotherapy
and autologous stem-cell transplant suggest that chemo-
therapy has the potential to increase the number of CTCs.
These studies are
summarised in the following sections.
Preclinical studies
A series of studies in preclinical metastasis models have
indicated that disruption of angiogenesis or induction
of damage to endothelial cells can enhance metastasis.
Two important studies showed that inhibition of angio-
genesis was able to reduce primary tumour growth152,153
;

however, by inducing an hypoxic environment in the
residual tumour, the therapy promoted invasion and
metastasis. Treatment of RIP1‑Tag2 mice, which develop
pancreatic tumours spontaneously, with a neutralising
antibody targeting VEGFR‑2 led to a reduction of pri-
mary tumour volume152
, but also resulted in an increase in
local invasion and distant metastasis. Similar results were
obtained using sunitinib, a small-molecule inhibitor of
both VEGFR and PDGFR152
. Ebos et al.153
demonstrated
a similar phenomenon in transplantable models of breast
cancer and melanoma using the angiogenesis inhibitors
sunitinib, sorafenib, and SU10944. Antiangiogenic thera-
pies for cancer have generally not proved to be as effective
in improving the survival of patients with cancer as was
originally hoped154
. One of the reasons for this limited
effectiveness could be that the beneficial effects of target-
ing the tumour vasculature are offset by unwanted effects
on tumour cell hypoxia, invasion, and metastasis.
Other targeted therapies can also promote metastasis
in preclinical models. Treatment of mice bearing pri-
mary tumours derived from injection of A-375 human
melanoma cells with the BRAF inhibitor vemurafenib,
resulted in the suppression of tumour growth. When a
small proportion of vemurafenib-resistant A-375 cells
were injected together with parental A-375 cells, however,
treatment with vemurafenib resulted in the vemurafenib-
sensitive cells secreting factors that promoted the growth
and metastasis of vemurafenib-
resistant tumours155
. The
secreted factors activated AKT signalling in resistant cells,
thereby driving their
continued growth and metastasis.
Individual cytotoxic chemotherapy agents (such as
cyclophosphamide) have also been shown to promote
metastasis under certain conditions. High-dose cyclo-
phosphamide is effective in inducing treatment responses
in some cancers, although this agent can also cause
immunosuppression and, thus, is used to manage graft-
versus-hostdisease156
.Conversely,lowdosesofcyclophos-
phamide can enhance antitumour immune responses in
animal models157
. Man and colleagues158
showed that,
in addition to causing primary tumour shrinkage, both
low-dose and high-dose cyclophosphamide promoted
metastasis of subcutaneously implanted lung adeno
carcinomas to the lung. Other studies have demonstrated
that an injection of cyclophosphamide 24h before intra
venous inoculation of fibrosarcoma-derived cells mark-
edly enhanced tumour-cell colonization of the lung159
.
Another example is the heat shock protein 90 inhibitor
17‑AAG, which inhibited the growth of primary breast
tumours, but also promoted metastasis to the bone by
enhancing osteoclast formation160
.
Clinical studies with chemotherapy
In patients with detectable CTCs, a reduction of CTC
numberswithchemotherapyisassociatedwithfavourable
responses to treatment and improved survival compared
withpatientswithoutadeclineinCTCs142,152,153
.Thechange
in CTC numbers might even constitute a surro
gate end
pointforchemotherapyresponsiveness151,161,162
.Inpatients
which CTC numbers increase steadily after chemother-
apy, the increase is probably caused by the release of CTCs
from progressive disease. Direct evidence for mobiliza-
tion of CTCs by chemotherapy in humans is scarce, but
provocative findings have been reported for high-dose
chemotherapy and autologous stem-cell transplantation
for the treatment of advanced-stage or high-risk solid
tumours. For instance, Brugger and colleagues163
studied
the outcomes of 46 patients with NSCLC, SCLC, or breast
cancer who underwent peripheral blood stem-cell har-
vesting and CTC analysis after treatment with the cyto-
toxic chemotherapy drugs VP‑16, ifosfamide, cisplatin
and G‑CSF (administered to mobilize blood progeni
tor
cells for subsequent harvesting). Of 42 patients with no
detectable CTCs at baseline, 13 patients had detectable
CTCs after chemotherapy. In the remaining four patients
(with detectable baseline CTCs), CTC numbers increased
after chemotherapy164
.
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Mobilization of CTCs by chemotherapy might be
irrelevant in patients who already have metastatic disease;
however, when chemotherapy is given as neoadjuvant
therapy (before planned curative surgery or radiother-
apy), with the intention of treating both the local disease
andoccultmetastasis,aparadoxicalmetastasis-promoting
effect of CTC mobilization could be clinically important.
Inhestern and colleagues164
studied CTCs in patients with
advanced-stage oral and oropharyngeal squamous-cell
carcinoma treated with neoadjuvant chemotherapy,
surgery, and postoperative radiotherapy. These investi
gators found that CTCs were detectable in about 80%
of patients at baseline and that CTC numbers declined
during chemotherapy in most patients. After surgery and
before the planned adjuvant radiotherapy, however, CTC
numbers had increased to above baselinelevels.Thecause
of this phenomenon is unknown but in these patients,
who had potentially curable disease, chemotherapy was
unable to eradicate CTCs — CTC mobilization should be
considered as a possible cause for this observation.
Clinical studies: biological therapies
In the study by Brugger et al.163
, the relative contributions
of chemotherapy and G‑CSF to CTC mobilization could
not be separated. In another study165
, cells identified as
epithelial because they expressed epithelial cell-adhesion
molecule(EpCAM),werefoundtocontaminateharvested
haematopoietic progenitor cells mobilized by G‑CSF
alone in patients with metastatic breast cancer before
high-dose chemotherapy and autologous stem-cell trans-
plantation. Thus, G‑CSF alone might also mobilize CTCs
from the bone marrow or other metastatic sites. High-
dose chemotherapy and stem-cell transplantation are
no longer part of the standard therapy used for patients
with epithelial cancers because of the negative results
of randomised clinical trials166
; thus, the contribution of
mobilized tumour cells to disease relapse after autologous
stem-cell
transplantation has not been established.
G‑CSF-based therapy is commonly administered
to patients to treat or prevent neutropenia caused by
myelosuppressive chemotherapy. A meta-analysis of 17
randomized controlled trials of patients receiving chemo-
therapy concluded that the mortality rate associated with
infection (P=0.018) and febrile neutropenia (P<0.001)
was significantly reduced by the administration of G‑CSF;
however, insufficient data were available to reach any
conclusions about the effect of G‑CSF on overall sur-
vival167
. In preclinical studies, we and others have shown
that the administration of G‑CSF promotes metastasis of
mammary tumours168,169
and cell migration induced by
low-dose radiation, for example, is dependent on G‑CSF
signalling in NSCLC cells62
. As well as acting on tumour
cells that express the G‑CSF receptor, G‑CSF can mobilize
myeloid cells that have potent immunosuppressive activ-
ity against T cells, thereby enhancing metastasis168
. The
implications of these findings in the treatment of patients
with cancer are unclear.
Erythropoietin is another haematopoietic growth
factor that has been used to treat patients with cancer.
Erythropoietin receptors can be found on the surface
of different cancer cell lines, leading to concerns about
a potential role in tumour growth stimulation170
. In a
randomised trial comparing erythropoietin plus radio
therapy with radiotherapy alone in patients with head and
neck cancer171
, the 5‑year estimate of locoregional failure
was 46.2% versus 39.4% (P=0.42); therefore, a detri
mental effect of erythropoietin was not ruled out by the
study investigators.
The publication of reports on the use of molecularly
targeted agents in the treatment of patients with cancer
has rapidly grown in the past few years. The mechanisms
of action of such agents are completely different from
those of conventional cytotoxic agents. No good clinical
evidence exists on the potential role of these agents in
CTC dissemination, but this possibility should be con-
sidered when evaluating long-term results of treatments
with molecularly targeted agents.
Systemic therapy–metastasis interactions
The possibility of a metastasis-promoting effect from
systemic therapy in patients with cancer is a challen
ging research question, especially because patients often
derive a benefit from such therapies. Nevertheless, seek-
ing evidence for such an effect is justifiable to improve
the overall benefits of antitumour therapies. A potential
cancer-
promoting effect derived from systemic cytotoxic
anticancer therapies seems paradoxical because of the
therapeutic nature of such therapies; if this effect does
occur,theimmediateclinicalcoursewouldbedetermined
by the pre-existing macroscopic tumour burden.
CTC mobilization by chemotherapy could poten-
tially be important when neoadjuvant chemotherapy
is used to treat locoregionally confined advanced-stage
tumours before administering curative-intent surgery or
radiotherapy and/or chemoradiotherapy. In patients with
macroscopic stage IIIA NSCLC, chemotherapy followed
bysurgerysubstantiallyimprovedsurvivalcomparedwith
surgery alone172
. By contrast, only a small improvement in
survival has been observed when surgery is followed by
adjuvant chemotherapy173
. In breast cancer, however, the
benefits of neoadjuvant and adjuvant chemotherapy are
similar174
. The interactions between therapeutic modal-
ities are complex, and likely to vary substantially across
cancer types.
The superior outcomes reported across a range of epi-
thelial cancers for concurrent versus sequential chemo-
radiotherapy are usually attributed solely to an effect on
local disease control, but accurate information on a direct
link between local disease control and systemic disease
can be difficult to obtain from data generated in clinical
trials. Most frequently, only the site of first failure (local
or systemic) is reported, and subsequent local or distant
failures are not described. Nevertheless, distant and local
treatment failure have been recorded separately and
analysed according to FDG-PET response in a study
involving patients with locoregionally advanced-stage
NSCLC treated with concurrent chemoradiotherapy175
.
A powerful negative correlation between metastasis and
a favourable local treatment response emerged from this
study, indicating that chemoradiotherapy-responsive
tumour cells were less likely to metastasise during or
after treatment175
. Concurrent chemoradiotherapy could
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reduce the risk of late distant relapse both by decreasing
the probability of spread from uncontrolled local disease,
and by ensuring that CTCs mobilized during treatment
have sustained more damage — and, therefore, would
be less capable of forming distant metastasis than CTCs

mobilized during radiotherapy as a single modality.
Conclusions
The relationships between frontline treatments, local
tumour control, and metastasis in patients with solid
tumours are complex and remain poorly understood.
Broad generalisations cannot be made from the studies
addressing these relationships because of the diversity
of cancers and antitumour therapies involved, as well as
variations in treatment delivery, CTC detection methods,
and time points analysed, together with the long time
period spanned by the publication of relevant observa-
tions. A common finding in these studies, however, is
that widely used antitumour therapies might be associ-
ated with an early increase in either the number or the
prolifera
tive capacity of CTCs. In studies analysing both
changes in CTC numbers and survival, an increase in
CTCs was in general associated with a less-beneficial out-
come. None of these studies has established a direct link
between metastasis and CTC mobilization by treatment,
but they do indicate an association between increased
CTC numbers after the initiation of therapy and an

elevated risk of metastasis.
The results of these studies do not imply that the cur-
rent development of antitumour treatment methods,
often the hard-won results of painstaking sequential ran-
domized trials, should be abandoned because they might
promote CTC mobilization. These treatments enable
many patients with locoregionally confined advanced-
stage solid tumours, who would otherwise suffer and
die from progressive disease, to be cured or have long-
term disease-free survival. The best available treatment
cannot be administered without a diagnosis, but, if CTC
mobilization by invasive medical interventions or ther-
apy represents a major cause of antitumour treatment
failure, then this subject is worthy of intensive study. In
the meantime, using the least invasive means of effective
diagnosis and treatment in patients with cancer seems the
most prudent option.
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Acknowledgements
We are grateful to David Ball for fruitful discussions and his
continuous support of our scientific initiatives, and to
Bernhard Riedel for his critical reading of the manuscript. We
thank Tim Akhurst for his assistance with Figure 3. O.A.M.
and M.P.M. receive support from the Australian National
Health and Medical Research Council (NHMRC) grant
1104139, and the Peter MacCallum Cancer Foundation
grant 1218. R.L.A. receives fellowship support from the
National Breast Cancer Foundation of Australia.
Author contributions
O.A.M., R.L.A. and M.P.M. researched data for the article,
and K.N. contributed to discussion of the article’s content. All
authors wrote, reviewed and edited the manuscript before
submission.
Competing interests statement
The authors declare no competing interests.
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