pantel ea - 2012 - circulating cancer cells and dna - v2

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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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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

200 Alix-Panabieres Schwarzenbach Pantel


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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.



12/20

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.



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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.

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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/

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/978-90-481-9381-3


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Annual Review

Medicine

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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

pantel ea - 2012 - circulating cancer cells and dna - v2

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