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Circulating Tumor Cells anCirculating Tumor DNA
Catherine Alix-Panabieres,1,2,3
Heidi Schwarzenbach,4 and Klaus Pantel4
1University Medical Center, Saint-Eloi Hospital, Institute of Research in BiotherapyLaboratory of Rare Human Circulating Cells, Montpellier, France; 2University MedCenter, Laboratory of Cell and Hormonal Biology, Arnaud de Villeneuve Hospital,
Montpellier, France; 3University Institute of Clinical Research UM1 EA2415 Epidemiology, Biostatistics & Public Health; email: c-panabieres@chu-montpellier.
4Institute of Tumor Biology, University Medical Center, Hamburg-Eppendorf,20246 Hamburg, Germany; email: [email protected]
Annu. Rev. Med. 2012. 63:199215
First published online as a Review in Advance on
November 2, 2011The Annual Review of Medicine is online atmed.annualreviews.org
This articles doi:10.1146/annurev-med-062310-094219
Copyright c 2012 by Annual Reviews.All rights reserved
0066-4219/12/0218-0199$20.00
Keywords
tumor cell dissemination, cell-free tumor DNA, allelic imbalance
genetic alterations, epigenetic alterations
Abstract
Solidtumors derived fromepithelial tissues(carcinomas) are responfor 90% of all new cancers in Europe, and the main four tumor
ties are breast, prostate, lung, and colon cancer. Present tumor stais mainly based on local tumor extension, metastatic lymph nod
volvement, and evidence of overt distant metastasis obtained by iing technologies. However, these staging procedures are not sen
enough to detect early tumor cell dissemination as a key event in tu
progression. Many teams have therefore focused on the developme
sensitive assays that allow the specific detection of single tumor cesmall amounts of cell-free tumor DNA in the peripheral blood ofcer patients. These methods allow the detection and characteriz
of early metastatic spread and will provide unique insights into thology of metastatic progression of human tumors, including the ef
of therapeutic interventions.
199
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REVIEWS
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Circulating tumorcells (CTCs): cellsshed by the primarytumor into thebloodstream very earlyin tumor development
Disseminated tumorcells (DTCs): CTCsthat have left the bloodcirculation and homedinto secondary organs
MRD: minimalresidual disease
BM: bone marrow
Circulating cell-freetumor DNA: DNAreleased by apoptoticand necrotic cells of
the primary tumorinto the bloodcirculation early intumor development
INTRODUCTION
Early during the formation and growth of aprimary tumor (e.g., breast, colon, or prostate
cancer), cells are shed from the primary tumorand circulate through the bloodstream. These
circulating tumor cells (CTCs) are very het-erogeneous and can be enriched and detected
via different technologies based on their physi-cal and biological properties. CTC analyses are
considered a real-time liquid biopsy in cancer
patients.The prognosis of carcinoma patients, even
with small primary tumors, is mainly deter-mined by the blood-borne dissemination of
tumor cells from the primary site to distantorgans such as bone marrow, liver, lungs, or
brain, and the subsequent outgrowth of thesecells in their new microenvironment (1, 2).
Disseminated tumor cells (DTCs) are consid-ered micrometastases. They can remain in a
dormant state for many years after completeresection of the primary tumor before giving
rise to macrometastasis (3, 4). DTCs recircu-
lating through the bloodstream may colonizeother distant organs, giving rise to secondary
metastases. Interestingly, DTCs can even re-turn to the primary tumor, a process termed
tumor self-seeding or cross-seeding, giving riseto aggressive metastatic variants. These DTCs
could thereby potentially contribute to the de-velopment of local relapses (5, 6), although this
provocative hypothesis requires support fromstudies in cancer patients.
Minimal residual disease (MRD), i.e., the
presence of DTCs, is undetectable by high-resolution imaging technologies. However,
DTCs can now be identified in the bone mar-row (BM), lymph nodes, or circulating blood,
using sensitive and specific assays (1, 4). BM iseasily accessible by needle aspiration through
the iliac crest, and it plays the most prominentroleamongthedistantorgansasindicatororgan
for MRD thus far. BM appears to be a commonhoming organ for DTCs derived from carcino-
mas of different organs (7) and also might bea reservoir for DTCs with the capacity to re-
enter other distant organs.
For the follow-up of cancer patients, s
quential analyses are pivotal. Because BM nedle aspiration is far more invasive than sam
pling of peripheral blood, research groups acurrently evaluating the clinical utility of t
mor cells in the blood rather than BM to asseprognosis and monitor systemic therapy (4).
number of innovative technologies to impromethods for CTC detection with extraordina
ily high sensitivity have recently been deveoped, including CTC microchips, filtration d
vices, quantitative RT-PCR assays, and aut
mated microscopic systems (1, 4). However, tspecificity and clinical utility of these metho
still have to be demonstrated in large prospetive multicenter studies to reach the high lev
of evidence required for introduction into cliical practice.
Concentrations of circulating cell-free tmor DNA are high in cancer patients com
pared to healthy individuals. Early in tumor dvelopment, apoptotic and necrotic cells of t
primary tumor release DNA into the bloo
stream (Figure 1). In the peripheral bloothis cell-free DNA circulates predominan
in the form of nucleosomes, indicating thit retains at least some features of the n
clear chromatin. This DNA can be extractfrom blood, and its genetic and epigenetic a
terations can be determined (8). Epigene
modifications include DNA methylation anconfiguration changes in chromatin histoproteins (9). In chromosomal regions of tumo
associated genes, epigenetic modifications maffect important regulatory mechanisms f
the pathogenesis of malignant transformatio
DNA methylation of the cytosine base in Cpdinucleotides, which are found as isolated
clustered CpG islands, induces gene represion by inhibiting the access of transcriptio
factors to their binding sites. Inactivation
tumor suppressor genes by promoter hypemethylation is thought to play a crucial role tumorigenesis.
However, the aberrations of the cell-frDNA in blood do not always match those
the primary tumor (8). This discrepancy m
be ascribed at least partly to CTCs or DTC
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CTC
+ DNA
Primary tumoror metastasis
Extraction ofcell-free DNA
b Capillary gel electrophoresis
c DNA sequencing
Blood
vesse
l
1
2
0
600
1,200
0150
LOH120 130
220 230
Wild type DNA
Microsatellite PCRusing cell-ree DNA
or detection o LOH
Methylation-sensitivePCR using bisulfte-converted cell-ree
DNA or detection omethylated DNA
Cell-ree blood DNA
Length of the PCR product (bp)
F
luorescence
intensity
600
1,200T TAT T T CG G GT CGAT T TAG AG
TGAG GGAT GAT TA AAGGTTTTTT
a
Figure 1
Detection of genetically and epigenetically altered DNA in blood. High levels of cell-free tumor DNA circulate in the blood of cpatients. (a) This tumor DNA found in blood can be released from either the primary tumor or (micro)metastasis, or apocirculating tumor cells (CTCs). This DNA can be extracted from blood, and the genetic and epigenetic alterations can be determTo detect loss of heterozygosity (LOH) on cell-free DNA, extracted DNA is amplified in a polymerase chain reaction (PCR)-bafluorescence microsatellite analysis using a gene-specific primer set binding to tumor suppressor genes. The fluorescence-labeledproducts can be separated by capillary gel electrophoresis and detected by a fluorescence laser. In the diagram ( b), the abscissa inthe length of the PCR product; the ordinate gives information on the fluorescence intensity represented as peaks. The upper anparts of the diagram show the PCR products derived from wild-type DNA (from leukocytes) and plasma DNA, respectively. Asdepicted by the two peaks of the amplified wild-type DNA, both alleles are intact, whereas the lower peak of the PCR product dfrom the plasma DNA shows LOH (arrow). (c) To detect cell-free methylated DNA, extracted DNA is denatured and treated wisodium bisulfite. In a methylation-sensitive PCR, the modified DNA is amplified with gene-specific primers. Because sodium biconverts unmethylated cytosine residues into uracil, in contrast to methylated cytosine, the methylation pattern can be determinDNA sequencing.
www.annualreviews.org Circulating Tumor Cells and Tumor DNA 201
Circulating cancer cell-freeDNA can be released from (1)he primary tumor or(micro)metastasis or (2)apoptotic circulating tumorcells (CTCs)
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which can also release their DNA into the blood
circulation (Figure 1).This review focuses on the detection and
further characterization of individual CTCsand circulating cell-free DNA as real-time liq-
uid biopsies that can help identify therapeutictargets and potential mechanisms of resistance
to therapy. This strategy might contribute tothe development of improved individualized
targeted treatment of cancer patients.
CIRCULATING TUMOR CELLS
Technologies Used forCTC Detection
The detection of CTCs in peripheral blood of
cancer patients holds great promise, and manyexciting technologies have been developed over
the past few years. However, detecting CTCsremains technically challenging. CTCs occur
at very low concentrations of one tumor cell
in the background of millions of blood cells.Their identification and characterization re-
quire extremely sensitive and specific analyticalmethods, which are usually a combination of
enrichment and detection procedures. Below,we briefly introduce some of the key tech-
nologies of CTC detection, although a com-prehensive overview of all existing assays and
publications would be beyond the scope of thisarticle.
CTC enrichment. CTC enrichment includesalargepaneloftechnologiesbasedonthediffer-
ent properties of CTCs that distinguish themfrom the surrounding normal hematopoietic
cells (Figure 2), including physical proper-
ties (size, density, electric charges, deformabil-ity) and biological properties (surface protein
expression, viability, and invasion capacity).
Physical properties have the advantage thatthey allow CTC separation without labeling.Methods based on physical properties include
density gradient centrifugation (Ficoll, Onco-Quick); filtration through special filters, e.g.,
theISET(IsolationbySizeofEpithelialTumorCells) (10, 11) or a novel three-dimensional
microfilter (12); a new versatile labelfr
biochip using the unique differences in size andeformability of cancer cells (larger and stiff
than blood cells) (13, 14); a microfluidic devicombining multi-orifice flow fractionati
(MOFF) and the dielectrophoretic (DEcell separation technique (15); and a diele
trophoretic fieldflow fractionation (DEFFF) device that allows isolation of viab
CTCs by different response to DEP due to dference in size and membrane properties (16
Biological properties are mainly used in im
munological procedureswith antibodiesagaineither tumor-associated antigens (positive s
lection) or the common leukocyte antigCD45 (negative selection). Immunomagne
systems target an antigen with an antibody this coupled to a magnetic bead, and the antige
antibody complex is subsequently isolated vexposure to a magnetic field. Positive selectio
is usually carried out with antibodies againthe epithelial cell adhesion molecule (EpCAM
and subsequent immunocytological detectio
of CTCs is performed with antibodies to ctokeratins,the intermediatefilaments of epith
lial cells (1). Among the current EpCAM-bastechnologies, the FDA-approved CellSearch
system has gained considerable attention ovthe past seven years (4) and is the gold sta
dard for all new CTC detection metho
(17, 18).At present, there is a focus on the develo
ment of microfluidic devices (chips), whi
can handle very small blood volumes. A mcrofluidic platform called a CTC-chip consi
of an array of anti-EpCAM antibody-coatmicroposts (1922). The high CTC counts
nonmetastatic cancer patients and the freque
detection of positive events in healthy controwarrant further investigations on the specifici
of this assay. Recently, a new CTC-chip call
Ephesia, which uses columns of biofunctioalized superparamagnetic beads self-assemblin a microfluidic channel onto an array
magnetic traps (23), and another microfluidsystem using high-throughput selection, en
meration, and electrokinetic manipulation low-abundance CTCs have been introduc
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Blood vessel
a Physical properties
Size/deformability Density Electric charges Marker proteins
Invasive capacity
EpCAM
CD45
CAM
CD45+
EpCAM+ EpCAM+
EpCAM+
CD45+
EpCAM+
MagSweeper
Positive Positive Mic
Positive
N
Ep
PositiveGlyco A
Fluo-CAM
b Biological properties
Leukocytes
RBC
CTC
+
+ + +
+ + +
+ + +
+
+++
+
Figure 2
Enrichment of circulating tumor cells (CTCs) from the peripheral blood of cancer patients is based on physical or biological proof CTCs. (a) Physical properties include size (membrane filter devices), deformability (microfluidic system in a chip), density (Ficentrifugation), and electric charge (dielectrophoresis). (b) Biological properties include expression of cell surface markers and incapacity. Cell surface markers include an epithelial cell adhesion molecule (EpCAM) for positive selection and CD45 for negativselection; anti-EpCAM or anti-CD45 antibodies conjugated with magnetic beads, used to enrich CTCs in a magnetic field; andanti-EpCAM antibodies on microposts or columns of nanobeads. Invasive capacity refers to adherence and invasion of fluorescematrix. Abbreviations: glyco A, glycophorin A (a 131-amino-acid protein present at the extracellular surface of the human red blcell); CAM, cell adhesion matrix; fluo-CAM, fluorescent cell adhesion matrix; RBC, red blood cells.
(24); validation of these assays is still ongoing.Microdevices can handle cell numbers and sam-
ple volumes at least 10 times smaller (
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Epithelial-mesenchymaltransition (EMT):trans-differentiation ofepithelial cells intomesenchymal cells,
often activated duringcancer invasion andmetastasis. Epithelialcells lose their cellularpolarity, and theirepithelial cellcell andcellmatrix adhesioncontacts areremodeled. E-cadherinand some cytokeratinsare downregulated;markers ofmesenchymal cells
such as vimentin andN-cadherin areupregulated
samples derived from patients with metastatic
disease, which contained 12 23 CEpC per9 ml of blood, whereas such cells were not
identified in any samples from healthy donors.Through the use of a 3D nanostructured
substrate or nanofly paper technologya silicon-nanowire (SiNW) array coated with
anti-EpCAM antibodiesCTCs can also becaptured efficiently (26). A specialized laser
scanning cytometer (MAINTRACTM) providesanother EpCAM-based approach, which com-
bines the cell-sorting speed of flow cytometry
with the power to analyze the morphologicalproperties of a single positive cell (27). As al-
most all patient samplesyield an extraordinarilyhigh number of CTCs with this method (50 to
3105 or three log units higher than observedwith the other approaches), careful validation
studies on the specificity of this technology arewarranted.
Importantly, a subset of epithelial can-cer cells is likely to undergo epithelial-
mesenchymal transition (EMT) prior to en-tering the peripheral circulation. EMT is a
key developmental program that is often ac-tivated during cancer invasion and metasta-
sis. It is thought to be linked to the gain of
cancer stem cell properties. During EMT, E-cadherin and some cytokeratins are downreg-
ulated, whereas markers of mesenchymal cells
such as vimentin and N-cadherin are upregu-lated. By undergoing this transition, tumor cellsmay escape detection by conventional methods
(28). Although there is an ongoing debate onthe relevance of EMT in cancer patients as op-
posed to experimental studies in model systems(29), recent work suggested that EMT might
particularly affect tumor cells with stem cell
like properties (30, 31). Therefore, in recentyears, there has been great interest in investi-
gating EMT markers in CTCs (32, 33). Inter-
estingly, it has been reported that the presenceof mesenchymal markers on CTCs more accu-rately predicted worse prognosis than the ex-
pression of cytokeratins alone, demonstratingthat current assays based on epithelial antigens
may miss the most aggressive CTC subpopula-tion (3436). Thus, there is an urgent need for
optimizing CTC detection methods throu
the inclusion of markers that are not repressduring EMT but still allow the analyst to disti
guish CTCs from the surrounding blood celFor example, vimentin, the mesenchymal inte
mediate filament frequently expressed in carcnoma cellsthathave undergone an EMT, is al
expressed in blood cells and thereforecannotused as a CTC marker.
Another way to overcome the problem false negative findings due to EMT-like CTC
is a functional cell separation method called t
collagen adhesion matrix (CAM) assay, whihas been reported in breast, prostate, and ova
ian cancer. CAM ingestion and epithelial immunostaining identifies CTCs based on the
invasive properties in vitro (37).Besides the choice of the appropriate CT
marker,thelimitedbloodsamplevolumesavaable from cancer patients may impose a ser
ous limitation on the detection of rare evensuch as CTCs. A completely new concept
enrichment and detection of CTCs has ther
fore been introduced and validated in a moumodel (38). As CTCs express folate and urok
nase plasminogen activator (uPA) receptothey can be dually targeted in the bloodstrea
with folate-conjugated nanotubes and manetic uPA-conjugated nanoparticles and subs
quently detected with two-color photoacous
flow cytometry. Future studies on humans wtell us whether this new platform can diagnotumor cell dissemination in cancer patients.
CTC detection. The CellSearch R syste
and the two CTC chips described above uthe same identification step: cells are fluore
cently stained for cytokeratins CK8, CK1
and CK19, the common leukocyte antigCD45, and a nuclear dye (4,6-diamino-
phenylindole; DAPI). Through multicolor im
age analysis with a fluorescent microscopCTCs are defined as CK+/CD45/DAPcells.
In order to detect only viable CTCs, thfunctional EPISPOT assay (for EPithelial Im
munoSPOT), which can be added to any kinof enrichment step, was introduced for CT
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Lung-
CTC
Bone-C
TC
Primary-
CTC
Primary-
CTC
Liver-CT
C
Primarydissemination
DTC
DTC
Distantorgans
Primary tumor Local relapse
Secondarydissemination
Blood
vesse
l
Blood
vesse
l
Figure 3
Editing of the circulating tumor cell (CTC) pool by the microenvironment of secondary metastatic sites.Tumor cells leave the primary tumor and circulate through the bloodstream. Each time the CTCs reach anew niche (distant organs, e.g., bone, liver, or lung in breast cancer), they undergo an organ-specificmimetism and may leave this site with a new organ-specific signature. Abbreviation: DTC, disseminatedtumor cell.
Osteomimetism:DTCs acquire bone-like properties to adapt
to the bonemicroenvironment,adopting anosteoblast-likephenotype and alteringthe functions ofosteoblasts andosteoclasts
clinical implications because niche signals mayregulate tumor dormancy and sensitivity tochemotherapy. In this context, it is important
to mention that DTCs established from BM ofcancer patients express a particular set of stress
proteins of the unfolded protein response thatenable cells to survive the hypoxic conditions
present in the niche areas and render them
more resistant to chemotherapy (49). More-over, a transcriptome analysis of osteotropic
breast tumor cells found in the BM has revealed
an osteoblast-like phenotype: these tumor cellsunderwent an osteomimetism in the bone byexpressing a pool of genes normally expressed
by osteoclasts or osteoblasts (5052). We canspeculate that such an adaptation may take
place each time a CTC reaches a new niche.Thus, it is conceivable that CTCs may acquire
an organ-mimetic phenotype, and CTexpressing lung-, bone-, and liver-specigenes after a particular niche adaptation mig
recirculate back into the blood (Figure 3).
Molecular Characterization of CTCs
Further characterization of CTCs is pivotal
provide insights into the complex biology tumor cell spread, with important implicatio
for defining therapeutic targets and elim
nating MRD. Because of the higher numbof detectable CTCs, the majority of resuconcerning genetic aberrations publish
thus far are derived from investigations CTCs from patients with castration-resista
prostate cancer (CRPC). Genotyping usioligonucleotide-array comparative genom
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hybridization revealed copy-number profiles
in CTCs from CRPC patients similar to thoseobserved in paired tumor tissues (53). By
multicolor fluorescence in situ hybridization(FISH), homogeneity in ERG oncogene
rearrangement status in CTCs from CRPCpatients was observed in contrast to significant
heterogeneity of androgen receptor (AR)copy-number gain and PTENloss (54). Swen-
nenhuis et al. reported that the majority ofCTCs in hormone-refractory prostate cancer
are aneuploid. Furthermore, heterogeneous
copy numbers of chromosomes 1, 7, 8, and 17were observed (55). In a considerable number
of CTC samples from patients with CRPC,amplifications of the AR gene locus could be
detected (56, 57). Very recently, mutations inAR were identified in CTC-enriched periph-
eral blood samples from CRPC patients byapplying Transgenomics WAVE R denaturing
high-performance liquid chromatographytechnology followed by direct sequencing
(58). These studies demonstrate that CTCsdetected with the CellSearch R system have
tumor-specific genomic characteristics butcan still show a marked genetic heterogeneity
depending on the genomic aberration analyzed.
Global gene expression profiles identifiedfor CTCs from patients with metastatic breast
cancer might be useful to distinguish normal
donors from cancer patients (59). One of thekey questions is whether CTCs exhibit a can-cer stem cell phenotype (see above). TWIST1,
a transcription factor pivotal for metastasis bypromoting EMT, was part of the gene expres-
sion signature identified in EpCAM-enrichedcells from BM of breast cancer patients af-
ter chemotherapy. TWIST1 expression was as-
sociated with distant metastasis and local tu-mor progression (60). Using the AdnaTest
TumorStemCell/AdnaTest EMT RT-PCT
assay, Aktas et al. also identified featurescharacteristic for stem cells and EMT in amajor proportion of CTCs from metastatic
breast cancer patients (61). A subpopulation ofCTCs with the putative stem cell phenotypes
CD44+/CD24/low or ALDH1high/CD24/low
was recently reported in a subpopulation of
patients with metastatic breast cancer using
triple-marker immunofluorescence microscopy(62). In a subset of breast cancer stem cells with
thepotential to self-renew, Notch seems to rep-resent a genetic biomarker that is frequently
coexpressed with the HER2 oncogene (63). In arecent study, Gazzaniga et al. identified a puta-
tive drug-resistance profile of CTCs with pre-dictive value for response to chemotherapy, in-
dependentofthetumortypeandthestageofthedisease, probably relevant for the individualiza-
tion of chemotherapy in cancer patients (64).
Clinical Relevance of CTC Analyses
Encouraging results concerning an associa-
tion between CTC detection and metastaticprogression in patients with metastatic breast,
prostate, and colorectal cancer have been re-cently published using the CellSearch R system
(56, 6570). Results indicated that CTCs in pe-ripheral blood of metastatic breast cancer pa-
tients at any time during therapy directly re-flect the patients response, or lack of response,
to therapy (71) and are therefore superior oradditive to conventional imaging methods (66,
72, 73). The randomized trial SWOG S0500,
led by the Southwest Oncology Group (74),which is expected to enroll 500 patients with
metastatic breast cancer, is now prospectively
addressing the clinical utility of CTC measure-ments in metastatic breast cancer patients. Thekey question for clinical use is whether the
change in therapeutic interventions based onCTC counts will result in a measurable benefit
(e.g., longer progression-free survival) for thecancer patient.
A challenging task for new techniques in-
tended to analyze CTCs/DTCs is to enabledetection and monitoring of MRD in patients
with nonmetastatic cancer. Recently, promis-
ing results derived from patients with non-metastatic breast cancer enrolled in neoadju-vant treatment studies. After an extension of
the median follow-up to 36 months, CTC de-tection before chemotherapy became an in-
dependent prognostic factor for both distantmetastasis-free survival and overall survival
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Loss ofheterozygosity(LOH): a commonform of allelicimbalance, which isused to identify
genomic regions thatharbor tumorsuppressor genes andto characterize tumorstages and progression
Microsatellite: ashort, highly repetitiveDNA sequence of 26base pairs occurring asa repetition of di-, tri-,or tetranucleotides. Inthe genome,microsatellites are
widely spread andusually lie innoncoding regions
(75). Interestingly, there was no significant cor-
relation between changes in CTC counts andresponse of the primary tumor to chemother-
apy, which is the current standard method toassess therapeutic efficacy in neoadjuvant ther-
apies. Thus, CTCs may have a different re-sponse pattern than primary tumor cells, and
measuring changes in CTC counts may pro-vide additional information on the response
of an individual patient. Follow-up analysesof two German trials in breast cancer us-
ing the CellSearch R technologythe GEPAR-
Quattro trial on neoadjuvant chemotherapyand additional trastuzumab treatment (if in-
dicated), and the SUCCESS trial on adjuvantchemotherapyare still ongoing and will show
whether the observed decreases in CTC rateswill be associated with an improved survival
rate (76, 77). As recently described by Xenidiset al., patients with detectable CK19 mRNA
post chemotherapy had a significantly reducedoverall and disease-free survival (78).
DTCs/CTCs capable of surviving chemo-therapy probably persist in a dormant, non-
proliferating state over many years (79, 80).
Targeted anticancer therapies that are moreeffective than current therapies and less harm-
ful to normal cells are being developed to killthese dormant cells specifically, or at least
block their outgrowth into overt metastases.
Currently, the choice of a targeted therapy foran individual patient is made upon analyzingthe primary tumor for the expression and/or
genomic status of a specific molecular target.However, this is often hampered by the hetero-
geneity and plasticity of individual tumor cellsin this tissue (81). Several studies have shown
that metastatic cells may have phenotypic and
genotypic characteristics that are distinct fromthe bulk of the primary tumor (82), which can
be explained by the facts that (a) the metastatic
subclone within the primary tumor might besmall and easily missed and (b) metastatic cellsmay gain additional genomic characteristics
over time and develop independently from theprimary tumor (83). Thus, the direct analysis
of metastatic cells may provide important addi-tional information for stratification of patients
to expensive therapies with considerable si
effects. For example, information regardithe expression of the estrogen receptor or th
HER2 oncogene on CTCs might be helpffor stratification and monitoring of endocri
therapy or therapy with trastuzumab (humanti-HER2 antibodies), which are curren
used in breast cancer only based on the analyof the primary tumors. However, an increasin
number of reports indicates a clear discrepanbetween the primary tumor status of these ta
gets and their expression on CTCs and DTC
(76, 8486).
CIRCULATING TUMOR DNA
Investigations on combined analyses of CTCand cell-free circulating tumor DNA have ju
begun and showed a potential relationship btween tumor DNA in blood serum/plasm
CTCs in blood, and DTCs in BM of cancer ptients (8792), suggesting circulating cell-fr
tumor DNA as a new biomarker for metastaspread in solid tumors.
In patients with primary head andneck squmous cell carcinoma, allelic imbalance, such
loss of heterozygosity (LOH), in serum DN
measured by a PCR-based fluorescence mcrosatellite analysis has for the first time be
reported to be helpful in identifying patients
risk for distant metastasis. Since these patienwith tumor DNA in their serum had a highrate of distantmetastasis,CTCs may contribu
to the presence of serum tumor DNA (89).In prostate cancer patients, the presen
of CTCs was detected by an epithelial immunospot assay and significantly correlat
with tumor stage, increasing Gleason scor
and frequencies of LOH at three markeencoding the cytoskeletal protein demat
the inhibitor of the cyclin-dependent kina
CDKN2/p16, and BRCA1 (90). These findinshowed a relationship between the presenof CTCs and the circulating tumor-associat
DNA in blood. Therefore, circulating cell-frtumor DNA might become a valuable sour
of information on metastatic progression prostate cancer and contribute to a bett
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understanding of the early steps of the
metastatic cascade in this carcinoma type.In breast cancer, CTCs and tumor-specific
alterations in cell-free plasma DNA were com-pared as markers for subclinical metastases. In
the study of Shaw et al., CTCs, DNA concen-tration/integrity, and evidence ofHER2 ampli-
fication were measured in patients with indeter-minate early or metastatic disease. CTCs and
plasma DNA analyses together helped to iden-tify patients with metastatic disease (91).
The presence of CTCs pointed not only
to genetic aberrations but also to epigeneticalterations of cell-free DNA. In stage IV
melanoma patients before administration ofbiochemotherapy, the number of CTCs de-
tected in blood significantly correlated with themethylation of cell-free RASSF1A and RAR-
2 DNA molecules (87). To assess the presenceof CTCs, a multimarker quantitative real-time
RT-PCR assay was performed in this study.Fordetectionof thecell-free, methylatedDNA,
the PCR products of sodium bisulfitemodifiedDNA were analyzed by capillary array elec-
trophoresis. CTCs and methylated RASSF1A
and RAR-2 on cell-free DNA were associ-ated with the outcome of patients. Patients with
CTCs and these methylated markers in theirblood showed significantly poorer response to
biochemotherapy and shorter time to progres-
sion and overall survival. These findings indi-cate that a combined assessment of cell-freemethylated DNA and CTCs in blood may be
a useful determinant of disease status and ef-ficacy of systemic therapy of melanoma (87).
Also, in breast cancer patients, the high in-cidence of cell-free methylated DNA corre-
lated with the occurrence of CTCs in the pe-
ripheral blood (92). Based on the relationshipof cell-free, methylated adenomatous polypo-
sis coli (APC), Ras association domain family
protein 1A (RASSF1A), and estrogen receptor1 (ESR1) DNA molecules with CTCs, it wasdeduced that CTCs are a potential source of
circulating tumor-specific DNA, and that highnumbers of CTCs and methylated DNA in
the blood are both indicators of more aggres-sive breast tumors (92). Moreover, in another
study, the detection of methylated APC and
glutathione s-transferase pi1 (GSTP1) DNA inserum of breast cancer patients correlated with
the presence of CTCs, which were measured bya modified immunomagnetic Adna test. Both
methylated DNA and CTCs correlated witha more aggressive tumor and advanced disease
(88).Taken together, blood may be a reservoir
collecting DNA from different sources, includ-ing CTCs and occult micrometastatic deposits
in secondary organs. Combining these DNA
analyses with the screening for CTCs mayprovide additional information for molecular
staging of tumors and monitoring of tumorprogression.
CONCLUSIONS
A considerable number of rare-cell detection
techniques have been developed during recentyears and are being continuously improved
by several working groups. DTCs in BM andof CTCs in blood of cancer patients can be
detected years before the occurrence of distantovert metastases. Nevertheless, analysis of
DTCs/CTCs is still not part of routine tumor
staging in clinical practice. This is mainly dueto the low number of these cells detectable with
the currently available methods, limiting their
value as a liquid biopsy especially in patientswith early-stage tumors. The new approacheshave to be evaluated for reproducibility,
sensitivity, and specificity in order to becomeapplicable for clinical practice. In addition, we
need to identify the most aggressive subset ofCTCs that are the metastasis-initiating cells
(93). Therefore, we need to develop better
strategies that are also able to isolate andidentify EMT-like subpopulations of tumor
cells with downregulated epithelial-specific
protein expression. Moreover, it might becomepossible to identify the tissue origin of CTCsby the detection of organ-specific metastatic
signatures in these cells using expressionprofiling, which would help to localize small,
occult metastatic lesions and guide furtherdiagnostic and therapeutic strategies.
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Information about CTC/DTC status may
be used to assess the individual prognosis ofcancer patients and to decide who should re-
ceivesystemictherapiesaimedtopreventrecur-rences and metastatic relapses. Furthermore,
CTC/DTC measurements within clinical tri-als might serve as important biomarkers for
real-time monitoring of the efficacy of systemictherapies in individual cancer patients, and
might thereby support accelerating drug devel-opmentanddefining subpopulations of patients
with the highest treatment benefit. Moreover,
CTC/DTC analysis for downstream compo-nents within signal transducing pathways that
influence new targeted therapies (e.g., KRASmutations in EGFR-targetedtherapies or PI3K
mutations in HER2-targeted therapies) mightprovide new insights into the complex mecha-
nisms of drug resistance.Tumor-associated nucleic acids may be
enriched in the blood of patients with tumors,and these cell-free nucleic acids may reflect
MRD. It is likely that at least part of thecell-free DNA found in the blood of cancer
patients is derived from CTCs or DTCs
present in secondary organs, which explai
the discrepancies between the genetic patterobserved in the blood and in the autologo
primary tumors. The investigation of tumospecific genetic and epigenetic profiles reli
on plasma and serum, which are easily obtainfor the development of clinical assays. Alo
or in combination with the analyses of CTCcirculating cell-free DNA assays might allo
monitoring of metastatic progression aidentification of mutations relevant for t
response of patients to targeted therapies (20
However, the analysis of circulating cell-frtumor DNA is also challenging and requir
stringent technical quality control measur(8).
Independent of the technical approach usethe key question to be addressed in clinic
intervention trials is how the assessment CTCs and/or circulating cell-free tumor DN
will guide therapy toward a more efficieelimination of metastatic cells. Detection an
eradication of metastatic cells at an earlitime point clearly has the potential to decreacancer mortality.
SUMMARY POINTS
1. CTCs can be enriched using extremely sensitive and specific technologies based on their
physical and biological properties.
2. CTCs are usually detected by immunocytochemical or RT-PCR-based assays for
epithelial-specific proteins or mRNA species. Cytokeratins are the most widely usedCTC markers.
3. CTCs can be characterized at the molecular level by detecting chromosomal anomalies(i.e., gain, loss, mutations of genes) or gene expression signatures.
4. EMT- and stem celllike phenotypes are two key characteristics of the more aggressivesubsets of CTCs.
5. Each time CTCs reach a new distant organ (e.g., bone, liver, lung), they may acquire an
organ-specific phenotype following particular niche adaptation.
6. CTCs are clinically relevant because they are (a) an independent prognostic factor incancer patients and (b) a predictive biomarker for treatment efficacy.
7. Circulating cell-free tumor DNA might become a biomarker for metastatic spread in
solid cancer. Blood is a compartment collecting tumor DNA shed by apoptotic/necrotictumor cells derived from the primary tumor as well as from occult (micro)metastatic
deposits, DTCs, and CTCs.
210 Alix-Panabieres Schwarzenbach Pantel
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FUTURE ISSUES
1. Future CTC assays need to be evaluated for reproducibility, sensitivity, and specificity
in order to become applicable for clinical practice. Better strategies that are also ableto isolate and detect EMT-like subsets of CTCs with downregulated epithelial-specific
protein expression must be developed.
2. Identifying the tissue origin of CTCs by the detection of organ-specific metastatic sig-
natures in these cells using expression profiling of single cells or CTC-enriched cellfractions may help clinicians in the future to localize small, occult metastatic lesions and
guide further diagnostic and therapeutic strategies.
3. Understanding new signal-transducing pathways at the CTC/DTC level should allow
the discovery of new targeted therapies and specific mechanisms of drug resistance.
4. Developing standardized methodologies for circulating cell-free DNA analyses and val-
idating these assays in large prospective clinical studies are indispensable to implementthis interesting approach in the clinical management of cancer patients.
DISCLOSURE STATEMENTK.P. has received a research grant and speakers honoraria from Veridex. C.A.P. has received
honoraria from Veridex.
LITERATURE CITED
1. Pantel K, Brakenhoff RH, Brandt B. 2008. Detection, clinical relevance, and specific biological properties
of disseminating tumour cells. Nat. Rev. Cancer8:32940
2. Hanahan D, Weinberg RA. 2011. Hallmarks of cancer: the next generation. Cell144:64674
3. Aguirre-Ghiso JA. 2007. Models, mechanisms and clinical evidence for cancer dormancy.Nat. Rev. Cancer
7:83446
4. Pantel K, Alix-Panabieres C, Riethdorf S. 2009. Cancer micrometastases. Nat. Rev. Clin. Oncol. 6:339515. Kim MY, Oskarsson T, Acharyya S, et al. 2009. Tumor self-seeding by circulating cancer cells. Cell
139:131526
6. Aguirre-Ghiso JA. 2010. On the theory of tumor self-seeding: implications for metastasis progression in
humans. Breast Cancer Res. 12:304
7. Pantel K, Brakenhoff RH. 2004. Dissecting the metastatic cascade. Nat. Rev. Cancer4:44856
8. A comprehens
summary of cell-
nucleic acids.
8. Schwarzenbach H, Hoon DS, Pantel K. 2011. Cell-free nucleic acids as biomarkers in cancer
patients. Nat. Rev. Cancer11:42637
9. Cedar H, Bergman Y. 2009. Linking DNA methylation and histone modification: patterns and paradigms.
Nat. Rev. Genet. 10:295304
10. Vona G, Sabile A, Louha M, et al. 2000. Isolation by size of epithelial tumor cells: a new method for the
immunomorphological and molecular characterization of circulating tumor cells. Am. J. Pathol. 156:57
6311. Pinzani P, Salvadori B, Simi L, et al. 2006. Isolation by size of epithelial tumor cells in peripheral blood
of patients with breast cancer: correlation with real-time reverse transcriptase-polymerase chain reaction
results and feasibility of molecular analysis by laser microdissection. Hum. Pathol. 37:71118
12. Zheng S, Lin HK, Lu B, et al. 2011. 3D microfilter device for viable circulating tumor cell (CTC)
enrichment from blood. Biomed. Microdevices13:20313
13. Tan SJ, Lakshmi RL, Chen P, et al. 2010. Versatile label free biochip for the detection of circulating
tumor cells from peripheral blood in cancer patients. Biosens. Bioelectron. 26:17015
www.annualreviews.org Circulating Tumor Cells and Tumor DNA 211
7/27/2019 Pantel EA - 2012 - Circulating Cancer Cells and DNA - V2
14/20
14. Bhagat AA, Hou HW, Li LD, et al. 2011. Pinched flow coupled shear-modulated inertial microfluid
for high-throughput rare blood cell separation. Lab Chip 11:187078
15. Moon HS, Kwon K, Kim SI, et al. 2011. Continuous separation of breast cancer cells from blood samp
using multi-orifice flow fractionation (MOFF) and dielectrophoresis (DEP). Lab Chip 11:111825
16. Gascoyne PR, Noshari J, Anderson TJ, et al. 2009. Isolation of rare cells from cell mixtures by diele
trophoresis. Electrophoresis30:138898
17. Andreopoulou E, Yang LY, Rangel KM, et al. 2011. Comparison of assay methods for detection
circulating tumor cells (CTCs) in metastatic breast cancer (MBC): AdnaGen AdnaTest BreastCanc
Select/Detect versus Veridex CellSearch system. Int. J. Cancer. Epub ahead of print18. Hofman V, Ilie MI, Long E, et al. 2011. Detection of circulating tumor cells as a prognostic factor
patients undergoing radical surgery for non-small cell lung carcinoma: comparison of the efficacy of t
CellSearch assay and the isolation by size of epithelial tumor cell method. Int. J. Cancer129:165160
19. Nagrath S, Sequist LV, Maheswaran S, et al. 2007. Isolation of rare circulating tumour cells in canc
patients by microchip technology. Nature 450:123539
20. Maheswaran S, Sequist LV, Nagrath S, et al. 2008. Detection of mutations in EGFR in circulating lun
cancer cells. N. Engl. J. Med. 359:36677
21. Stott SL, Lee RJ, Nagrath S, et al. 2010. Isolation and characterization of circulating tumor cells fro
patients with localized and metastatic prostate cancer. Sci. Transl. Med. 2:25ra23
22. Stott SL, Hsu CH, Tsukrov DI, et al. 2010. Isolation of circulating tumor cells using a microvorte
generating herringbone-chip. Proc. Natl. Acad. Sci. USA 107:1839297
23. Saliba AE, Saias L, Psychari E, et al. 2010. Microfluidic sorting and multimodal typing of cancer cells
self-assembled magnetic arrays. Proc. Natl. Acad. Sci. USA 107:1452429
24. Dharmasiri U, Njoroge SK, Witek MA, et al. 2011. High-throughput selection, enumeration, electro
netic manipulation, and molecular profiling of low-abundance circulating tumor cells using a microflui
system. Anal. Chem. 83:23019
25. Talasaz AH, Powell AA, Huber DE, et al. 2009. Isolating highly enriched populations of circulati
epithelial cells and other rare cells from blood using a magnetic sweeper device. Proc. Natl. Acad. Sci. US
106:397075
26. Wang S, Owens GE, Tseng HR. 2011. Nano fly paper technology for the capture of circulating tum
cells. Methods Mol. Biol. 726:14150
27. Pachmann K, Camara O, Kavallaris A, et al. 2008. Monitoring the response of circulating epithelial tum
cells to adjuvant chemotherapy in breast cancer allows detection of patients at risk of early relapse. J. Cl
Oncol. 26:120815
28. Muller V, Alix-Panabieres C, Pantel K. 2010. Insights into minimal residual disease in cancer patienimplications for anti-cancer therapies. Eur. J. Cancer46:118997
29. Ledford H. 2011. Cancer theory faces doubts. Nature 472:273
30. Mani SA, Guo W, Liao MJ, et al. 2008. The epithelial-mesenchymal transition generates cells wi
properties of stem cells. Cell133:70415
31. Mego M, Mani SA, Cristofanilli M. 2010. Molecular mechanisms of metastasis in breast cancerclini
applications. Nat. Rev. Clin. Oncol. 7:693701
32. Mego M, Mani SA, Lee BN, et al. 2011. Expression of epithelial-mesenchymal transition-inducing tra
scription factors in primary breast cancer: the effect of neoadjuvant therapy. Int. J. Cancer. Epub ahead
33. Hou JM, Krebs M, Ward T, et al. 2011. Circulating tumor cells as a window on metastasis biology
lung cancer. Am. J. Pathol. 178:98996
34. Gradilone A, Raimondi C, Nicolazzo C, et al. 2011. Circulating tumor cells lacking cytokeratin in brecancer: the importance of being mesenchymal. J. Cell Mol. Med. 15:106670
35. Raimondi C, Gradilone A, Naso G, et al. 2011. Epithelial-mesenchymal transition and stemness featu
in circulating tumor cells from breast cancer patients. Breast Cancer Res. Treat. 130:44955
36. Konigsberg R, Obermayr E, Bises G, et al. 2011. Detection of EpCAM positive and negative circulati
tumor cells in metastatic breast cancer patients. Acta Oncol. 50:70010
37. Lu J, Fan T, Zhao Q, et al. 2010. Isolation of circulating epithelial and tumor progenitor cells with
invasive phenotype from breast cancer patients. Int. J. Cancer126:66983
212 Alix-Panabieres Schwarzenbach Pantel
7/27/2019 Pantel EA - 2012 - Circulating Cancer Cells and DNA - V2
15/20
38. Galanzha EI, Shashkov EV,KellyT, et al. 2009. In vivo magnetic enrichment andmultiplex photoacoustic
detection of circulating tumour cells. Nat. Nanotechnol. 4:85560
39. First demons
that cytokeratins
released by viabl
and used as mark
EPISPOT assay.
39. Alix-Panabieres C, Vendrell JP, Slijper M, et al. 2009. Full-length cytokeratin-19 is released by
human tumor cells: a potential role in metastatic progression of breast cancer. Breast Cancer Res.
11:R39
40. Alix-Panabieres C, Vendrell JP, Pelle O, et al. 2007. Detection and characterization of putative metastatic
precursor cells in cancer patients. Clin. Chem. 53:53739
41. Krivacic RT, Ladanyi A, Curry DN, et al. 2004. A rare-cell detector for cancer. Proc. Natl. Acad. Sci. USA
101:10501442. Somlo G, LauSK, Frankel P, et al. 2011. Multiple biomarkerexpression on circulating tumor cells in com-
parison to tumor tissues from primary and metastatic sites in patients with locally advanced/inflammatory,
and stage IV breast cancer, using a novel detection technology. Breast Cancer Res. Treat. 128:15563
43. Ntouroupi TG, Ashraf SQ, McGregor SB, et al. 2008. Detection of circulating tumour cells in peripheral
blood with an automated scanning fluorescence microscope. Br. J. Cancer99:78995
44. Deng G, Herrler M, Burgess D,et al.2008.Enrichment with anti-cytokeratin alone orcombinedwithanti-
EpCAM antibodies significantly increases the sensitivity for circulating tumor cell detection in metastatic
breast cancer patients. Breast Cancer Res. 10:R69
45. Scholtens TM, Schreuder F, Ligthart ST, et al. 2011. CellTracks TDI: An image cytometer for cell
characterization. Cytometry A. 79:20313
46. Markou A, Strati A, Malamos N, et al. 2011. Molecular characterization of circulating tumor cells in breast
cancer by a liquid bead array hybridization assay. Clin. Chem. 57:4213047. Agrawal B, Krantz MJ, Parker J, et al. 1998. Expression of MUC1 mucin on activated human T cells:
implications for a role of MUC1 in normal immune regulation. Cancer Res. 58:407981
48. Schuettpelz LG, Link DC. 2011. Niche competition and cancer metastasis to bone. J. Clin. Invest.
121:125355
49. Experimenta
shows that prost
tumor cells comp
the same niches
hematopoietic st
cells in the bone
marrow.
49. Shiozawa Y, Pedersen EA, Havens AM, et al. 2011. Human prostate cancer metastases target
the hematopoietic stem cell niche to establish footholds in mouse bone marrow. J. Clin. Invest.
121:1298312
50. Bellahcene A, Bachelier R, Detry C, et al. 2007. Transcriptome analysis reveals an osteoblast-like pheno-
type for human osteotropic breast cancer cells. Breast Cancer Res. Treat. 101:13548
51. Garcia T, Jackson A, Bachelier R, et al. 2008. A convenient clinically relevant model of human breast
cancer bone metastasis. Clin. Exp. Metastasis25:3342
52. Le Gall C, Bellahcene A, Bonnelye E, et al. 2007. A cathepsin K inhibitor reduces breast cancer induced
osteolysis and skeletal tumor burden. Cancer Res. 67:9894902
53. Paris PL, Kobayashi Y, Zhao Q, et al. 2009. Functional phenotyping and genotyping of circulating tumor
cells from patients with castration resistant prostate cancer. Cancer Lett. 277:16473
54. Attard G, Swennenhuis JF, Olmos D, et al. 2009. Characterization of ERG, AR and PTEN gene status
in circulating tumor cells from patients with castration-resistant prostate cancer. Cancer Res. 69:291218
55. Swennenhuis JF, Tibbe AG, Levink R, et al. 2009. Characterization of circulating tumor cells by fluores-
cence in situ hybridization. Cytometry A 75:52027
56. Shaffer DR, Leversha MA, Danila DC, et al. 2007. Circulating tumor cell analysis in patients with pro-
gressive castration-resistant prostate cancer. Clin. Cancer Res. 13:202329
57. Leversha MA, Han J, Asgari Z, et al. 2009. Fluorescence in situ hybridization analysis of circulating tumor
cells in metastatic prostate cancer. Clin. Cancer Res. 15:20919758. Jiang Y, Palma JF, Agus DB, et al. 2010. Detection of androgen receptor mutations in circulating tumor
cells in castration-resistant prostate cancer. Clin. Chem. 56:149295
59. Smirnov DA,Foulk BW,Doyle GV,et al.2006. Globalgene expression profiling of circulating endothelial
cells in patients with metastatic carcinomas. Cancer Res. 66:291822
60. Watson MA, Ylagan LR, Trinkaus KM, et al. 2007. Isolation and molecular profiling of bone marrow
micrometastases identifies TWIST1 as a marker of early tumor relapse in breast cancer patients. Clin.
Cancer Res. 13:50019
www.annualreviews.org Circulating Tumor Cells and Tumor DNA 213
7/27/2019 Pantel EA - 2012 - Circulating Cancer Cells and DNA - V2
16/20
61. Aktas B, Tewes M, Fehm T, et al. 2009. Stem cell and epithelial-mesenchymal transition markers a
frequently overexpressed in circulating tumor cells of metastatic breast cancer patients. Breast Cancer R
11:R46
62. Theodoropoulos PA, Polioudaki H, Agelaki S, et al. 2010. Circulating tumor cells with a putative ste
cell phenotype in peripheral blood of patients with breast cancer. Cancer Lett. 288:99106
63. Reuben JM, Lee BN, Li C, et al. 2010. Circulating tumor cells and biomarkers: implications for perso
alized targeted treatments for metastatic breast cancer. Breast J. 16:32730
64. Gazzaniga P, Naso G, Gradilone A, et al. 2010. Chemosensitivity profile assay of circulating cancer ce
prognostic and predictive value in epithelial tumors. Int. J. Cancer126:24374765. First study
demonstrating
convincing data for the
prognostic relevance of
CTCs in breast cancer
patients.
65. Cristofanilli M, Budd GT, Ellis MJ, et al. 2004. Circulating tumor cells, disease progression, a
survival in metastatic breast cancer. N. Engl. J. Med. 351:78191
66. Cohen SJ, Punt CJ, Iannotti N, et al. 2008. Relationship of circulating tumor cells to tumor respon
progression-free survival, and overall survival in patients with metastatic colorectal cancer. J. Clin. Onc
26:321321
67. de Bono JS, Attard G, Adjei A, et al. 2007. Potential applications for circulating tumor cells expressi
the insulin-like growth factor-I receptor. Clin. Cancer Res. 13:361116
68. First study
demonstrating that
CTC counts are
complementary to
serum markers for
measuring therapyresponse.
68. de Bono JS, Scher HI, Montgomery RB, et al. 2008. Circulating tumor cells predict survival bene
from treatment in metastatic castration-resistant prostate cancer. Clin. Cancer Res. 14:63029
69. Danila DC,Heller G, Gignac GA, et al. 2007. Circulating tumor cell numberand prognosis in progress
castration-resistant prostate cancer. Clin. Cancer Res. 13:705358
70. Liu MC, Shields PG, Warren RD, et al. 2009. Circulating tumor cells: a useful predictor of treatme
efficacy in metastatic breast cancer. J. Clin. Oncol. 27:515359
71. Hayes DF, Cristofanilli M, Budd GT, et al. 2006. Circulating tumor cells at each follow-up time poi
during therapy of metastatic breast cancer patients predict progression-free and overall survival. Cl
Cancer Res. 12:421824
72. Budd GT,Cristofanilli M, Ellis MJ,et al.2006. Circulating tumor cells versus imagingpredicting over
survival in metastatic breast cancer. Clin. Cancer Res. 12:64039
73. De Giorgi U, Valero V, Rohren E, et al. 2009. Circulating tumor cells and [18F]fluorodeoxygluco
positron emission tomography/computed tomography for outcome prediction in metastatic breast canc
J. Clin. Oncol. 27:330311
74. National Cancer Institute. 2011. Treatment decision making based on blood levels of tumor ce
in women with metastatic breast cancer receiving chemotherapy. SWOG-S0500. http://clinicaltria
gov/ct2/show/NCT00382018
75. Bidard FC, Mathiot C, Delaloge S, et al. 2010. Single circulating tumor cell detection and overall surviin nonmetastatic breast cancer. Ann. Oncol. 21:72933
76. Study develops
scoring system for
HER2 status of CTCs
and demonstratesstriking heterogeneity
of CTCs and primary
breast tumors.76. Riethdorf S, Muller V, Zhang L, et al. 2010. Detection and HER2 expression of circulating tum
cells prospective monitoring in breast cancer patients treated in the neoadjuvant GeparQuatt
Trial. Clin. Cancer Res. 16:263445
77. Rack B, Schindlbeck C, Andergassen U, et al. 2010. Use of circulating tumor cells (CTC) in periphe
blood of breast cancer patients before and after adjuvant chemotherapy to predict risk for relapse: t
SUCCESS Trial. J. Clin. Oncol. 28:7s
78. Study shows that
CTCs that survive
chemotherapy are
indicators of poor
prognosis in breast
cancer.
78. Xenidis N, Ignatiadis M, Apostolaki S, et al. 2009. Cytokeratin-19 mRNA-positive circulati
tumor cells after adjuvant chemotherapy in patients with early breast cancer. J. Clin. Onc
27:217784
79. Study shows
significant correlation
between detection of
CTCs and DTCs in
bone marrow, as well as
low proliferative activity
of CTCs.
79. Muller V, Stahmann N, Riethdorf S, et al. 2005. Circulating tumor cells in breast cancer: co
relation to bone marrow micrometastases, heterogeneous response to systemic therapy and loproliferative activity. Clin. Cancer Res. 11:367885
80. Pantel K, Schlimok G, Braun S, et al. 1993. Differential expression of proliferation-associated molecu
in individual micrometastatic carcinoma cells. J. Natl. Cancer Inst. 85:141924
81. Stoecklein NH, Klein CA. 2010. Genetic disparity between primary tumours, disseminated tumour ce
and manifest metastasis. Int. J. Cancer126:58998
82. PantelK, Alix-PanabieresC. 2010.Circulating tumour cellsin cancer patients:challenges and perspectiv
Trends Mol. Med. 16:398406
214 Alix-Panabieres Schwarzenbach Pantel
http://clinicaltrials.gov/ct2/show/NCT00382018http://clinicaltrials.gov/ct2/show/NCT00382018http://clinicaltrials.gov/ct2/show/NCT00382018http://clinicaltrials.gov/ct2/show/NCT003820187/27/2019 Pantel EA - 2012 - Circulating Cancer Cells and DNA - V2
17/20
83. Klein CA. 2009. Parallel progression of primary tumours and metastases. Nat. Rev. Cancer9:30212
84. Fehm T, Hoffmann O, Aktas B, et al. 2009. Detection and characterization of circulating tumor cells in
blood of primary breast cancer patients by RT-PCR and comparison to status of bonemarrowdisseminated
cells. Breast Cancer Res. 11:R59
85. Braun S, Schlimok G, Heumos I, et al. 2001. ErbB2 overexpression on occult metastatic cells in bone
marrow predicts poor clinical outcome of stage IIII breast cancer patients. Cancer Res. 61:189095
86. Fehm T, Muller V, Aktas B, et al. 2010. HER2 status of circulating tumor cells in patients with metastatic
breast cancer: a prospective, multicenter trial. Breast Cancer Res. Treat. 124:40312
87. Koyanagi K, Mori T, ODay SJ,et al.2006.Association of circulating tumor cells with serumtumor-relatedmethylated DNA in peripheral blood of melanoma patients. Cancer Res. 66:611117
88. Matuschek C, Bolke E, Lammering G, et al. 2010. Methylated APC and GSTP1 genes in serum DNA
correlate with the presence of circulating blood tumor cells and are associated with a more aggressive and
advanced breast cancer disease. Eur. J. Med. Res. 15:27786
89. Nawroz-Danish H, EisenbergerCF, YooGH, et al.2004.Microsatelliteanalysisof serumDNA in patients
with head and neck cancer. Int. J. Cancer111:96100
90. For the first t
relationship betw
the occurrence o
and circulating tu
associated DNA
blood is describe
prostate cancer p
90. Schwarzenbach H, Alix-Panabieres C, Muller I, et al. 2009. Cell-free tumor DNA in blood plasma
as a marker for circulating tumor cells in prostate cancer. Clin. Cancer Res. 15:103238
91. Shaw JA, Brown J, Coombes RC, et al. 2011. Circulating tumor cells and plasma DNA analysis in patients
with indeterminate early or metastatic breast cancer. Biomark Med. 5:8791
92. Van derAuwera I, Elst HJ,Van LaereSJ, etal. 2009. Thepresence of circulating totalDNA andmethylated
genes is associated with circulating tumour cells in blood from breast cancer patients. Br. J. Cancer100:127786
93. Wicha MS, Hayes DF. 2011. Circulating tumor cells: not all detected cells are bad and not all bad cells
are detected. J. Clin. Oncol. 29:150811
RELATED RESOURCES
1. Circulating Nucleic Acids in Plasma and Serum. Proc. Int. Conf. Circulating Nucleic Acids
in Plasma and Serum, 6th, Hong Kong, Nov. 911, 2009. http://www.springer.com/biomed/cancer/book/978-90-481-9381-3
2. Website of the Institute of Tumor Biology, UKE, Hamburg. http://www.uke.uni-hamburg.de/institute/tumorbiologie
3. Website of the EU sixth FP Network Group DISMAL. http://www.dismal-project.eu/
www.annualreviews.org Circulating Tumor Cells and Tumor DNA 215
http://www.springer.com/biomed/cancer/book/978-90-481-9381-3http://www.springer.com/biomed/cancer/book/978-90-481-9381-3http://www.uke.uni-hamburg.de/institute/tumorbiologiehttp://www.uke.uni-hamburg.de/institute/tumorbiologiehttp://www.dismal-project.eu/http://www.dismal-project.eu/http://www.uke.uni-hamburg.de/institute/tumorbiologiehttp://www.uke.uni-hamburg.de/institute/tumorbiologiehttp://www.springer.com/biomed/cancer/book/978-90-481-9381-3http://www.springer.com/biomed/cancer/book/978-90-481-9381-37/27/2019 Pantel EA - 2012 - Circulating Cancer Cells and DNA - V2
18/20
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Andrew J. Vickers, Monique J. Roobol, and Hans Lilja p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 161
Targeting Metastatic Melanoma
Keith T. Flaherty p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 171
Nanoparticle Delivery of Cancer Drugs
Andrew Z. Wang, Robert Langer, and Omid C. Farokhzad p p p p p p p p p p p p p p p p p p p p p p p p p p p p 185
v
7/27/2019 Pantel EA - 2012 - Circulating Cancer Cells and DNA - V2
19/20
7/27/2019 Pantel EA - 2012 - Circulating Cancer Cells and DNA - V2
20/20
Eosinophilic Esophagitis: Rapidly Advancing Insights
J. Pablo Abonia and Marc E. Rothenberg p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 421
Physician Workforce Projections in an Era of Health Care Reform
Darrell G. Kirch, Mackenzie K. Henderson, and Michael J. Dill p p p p p p p p p p p p p p p p p p p p p p p 435
Reducing Medical Errors and Adverse Events
Julius Cuong Pham, Monica S. Aswani, Michael Rosen, HeeWon Lee,
Matthew Huddle, Kristina Weeks, and Peter J. Pronovostp p p p p p p p p p p p p p p p p p p p p p p p p p p p
447
Relationships Between Medicine and Industry: Approaches to the
Problem of Conflicts of Interest
Raymond Raad and Paul S. Appelbaum p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 465
Wireless Technology in Disease Management and Medicine
Gari D. Clifford and David Clifton p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 479
Geographic Variation in Health Care
Tom Rosenthal p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 493
Deep Brain Stimulation for Intractable Psychiatric DisordersWayne K. Goodman and Ron L. Alterman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 511
Contemporary Management of Male Infertility
Peter J. Stahl, Doron S. Stember, and Marc Goldstein p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 525
Indexes
Cumulative Index of Contributing Authors, Volumes 5963 p p p p p p p p p p p p p p p p p p p p p p p p p p p 541
Cumulative Index of Chapter Titles, Volumes 5963 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 545
Errata
An online log of corrections to Annual Review of Medicine articles may be found at
http://med.annualreviews.org/errata.shtml
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