[Advances in Clinical Chemistry] Volume 61 || Markers of Circulating Breast Cancer Cells

CHAPTER SIX

Markers of Circulating BreastCancer CellsEunice López-Muñoz*,1, Manuel Méndez-Montes†*Departamento de Genetica Medica, Unidad Medica de Alta Especialidad, Hospital de Gineco Obstetricia,No. 4, Dr. Luis Castelazo Ayala, IMSS, Colonia Tizapan San Angel, Mexico, D.F.†Departamento de Diseno Grafico, UnidadMedica de Alta Especialidad, Hospital de GinecoObstetricia, No. 4,Dr. Luis Castelazo Ayala, IMSS, Colonia Tizapan San Angel, Mexico, D.F.1Corresponding author: e-mail address: [email protected]

Contents

1.

AdvISShttp

Metastasis Cascade

ances in Clinical Chemistry, Volume 61 # 2013 Elsevier Inc.N 0065-2423 All rights reserved.://dx.doi.org/10.1016/B978-0-12-407680-8.00007-5

176
1.1 Local invasion of the ECM 178 1.2 Tumor cell migration 180 1.3 Epithelial–mesenchymal transition 181 1.4 Invadopodia 182 1.5 Intravasation 183 1.6 Circulation 184 1.7 Arrest in distant organs 185 1.8 Extravasation 185 1.9 Colonization 186 1.10 Metastatic dormancy 186
2.
Techniques for CTC Detection 187 2.1 Enrichment or isolation methods 188 2.2 CTC identification 197
3.
Molecular and Genetic Characterization 200 3.1 IHC 200 3.2 FISH 200 3.3 Multicolor FISH 201 3.4 CGH 201 3.5 Array CGH 202 3.6 Multimarker quantitative real-time RT-PCR 202 3.7 AmpliGrid 202 3.8 RNA in situ hybridization 202 3.9 Microarray 204
4.
Markers 205 5. Clinical Utility 207
5.1
Early breast cancer 207 5.2 Metastatic breast cancer 208
6.
Conclusion 209 References 210
175

http://dx.doi.org/10.1016/B978-0-12-407680-8.00007-5

176 Eunice López-Muñoz and Manuel Méndez-Montes

Abstract

The detection of circulating tumor cells (CTC) aids in diagnosis of disease, prognosis,disease recurrence, and therapeutic response. The molecular aspects of metastasisare reviewed including its relevance in the identification and characterization of puta-tive markers that may be useful in the detection thereof. Also discussed are methods forCTC enrichment using molecular strategies. The clinical application of CTC in the met-astatic disease process is also summarized.

1. METASTASIS CASCADE

Malignant neoplastic cells are capable of metastasis. Metastasis starts as

a small group of cells (usually not identified during routine histopathologic

staging) that spread from the site of primary origin.

Approximately 5% of patients with breast cancer have clinically detect-

able metastases at diagnosis, but 30–40% have occult metastases [1]. The for-

mation of metastatic clones begins early in primary tumor development.

To achieve successful metastasis, a cell group must be able to invade, survive,

and proliferate. This process requires cells to enter into circulation, stop in

the distal vascular bed, extravasate into the interstitium and parenchyma of

distant organs, and proliferate as a secondary colony.

Most cancer cells in the primary tumor have a metastatic phenotype [2].

This finding suggests that tumor cell spread likely occurs early in carcinogen-

esis [3]. Furthermore, it has been proposed that the same genes that cause the

primary tumor are involved in metastasis [2].

There are two scenarios of cancer progression. In the linear progression

model, neoplastic cells acquire a number of genetic, epigenetic, and mor-

phologic alterations. These changes allow migration across the basement

membrane (BM) and the extracellular matrix (ECM). Cells then enter

and travel via the circulatory or lymphatic system to a new site. In the parallel

progression model, tumor cell dissemination occurs at early disease stages,

that is, years before the diagnosis of a primary tumor, with tumor cell clones

adapted to the new microenvironment [4–10] (Fig. 6.1).

In traditional metastasis, progression is considered unidirectional in

which disseminated tumor cells (DTC) and circulating tumor cells

(CTC) are potentially initiating. They are derived from a small population

of primary tumor cells and increase their numbers at later stages of

progression [10].

Figure 6.1 Cancer progression. (A) Linear progression model, unidirectional. (B) Parallelprogression model, multidirectional.

177Circulating Markers of Breast Cancer

However, there is evidence that CTC can not only migrate multi-

directionally and seed in regional and distant sites of the body, but also return

to the original site of the primary tumor, a process called “tumor self-

seeding” [11,12]. In this process, more aggressive cell populations are likely

selected due to their ability to survive in the circulation. Attraction signals

result in reinfiltration of the primary tumor mass with relative ease due to a

leaky neovasculature. No additional adjustment to the receptor microenvi-

ronment is required [12].

Factors that determine the dissemination of tumor cells in the lymphatic

or hematogenous systems are not precisely known, nor is it clear whether

they are independent of each other. Recently, Hartkopf et al. [13] showed

that the spread of tumor cells by the hematogenous route was independent of

lymphatic dissemination. Phenotypic differences permitted discrimination

of cells with high and low metastatic potential and determined the preferred

direction of dissemination. The decision to intravasate lymphatic or blood

vessels may depend on physical limitations of the primary tumor. Lymph

vessels, unlike blood vessels, have a discontinuous basal membrane and lack

tight interendothelial junctions as well as surrounding pericytes/smooth

Figure 6.2 Tumor cell dissemination. (A) Lymphatic pathway permits detection of DTC.(B) Hematogenous pathway permits detection of CTC.


muscle cells. As such, access to these vessels influences the route of tumor

spread [13] (Fig. 6.2).

Irrespective of dissemination route, the metastatic process includes local

invasion of the ECM, intravasation, bloodstream or lymphatic survival,

arrest at distant organ sites, extravasation in tissue parenchyma, survival in

a foreign microenvironment, proliferation mechanisms to reset to the initial

formation of micrometastasis, and subsequent metastatic colonization [14]

(Fig. 6.3).

1.1. Local invasion of the ECMMutations in genes that encode proteins involved in cell–matrix and cell–

cell adhesion, as well as changes in the ECM and tumor microenvironment

enable local invasion [15–19].

The ECM is composed of proteins, glycoproteins, proteoglycans, and

polysaccharides [20–22]. These components also form the BM, which

Figure 6.3 Metastasis cascade: Hematogenous pathway.


separates the epithelium or stroma endothelium and the interstitial matrix.

The BM is a compact and specialized ECM with little porosity.

The ECM also possesses direct or indirect signaling properties that

allow cells to interact with their microenvironment via signal transduction

processes, which also influence gene expression [22]. The ECM also has bio-

mechanical properties, such as elasticity, which determines how cells mon-

itor and perceive external forces. The cytoskeleton, nuclear matrix, nuclear

membrane, and chromatin constitute the mechanosensory machinery that

determines cell reaction to ECM forces [22].

Alterations in structure, signal transduction or biomechanical properties

of the ECM have important roles in cancer development. In local invasion,

cancer cells disseminate to stromal and normal tissues surrounding the


primary tumor to induce BM thinning that potentially leads to rupture.

Invasion, migration, and dissemination can be random or directed. Directed

cell migration includes chemotaxis, haptotaxis, electrotaxis, and durotaxis

[23,24].

1.2. Tumor cell migrationMigration through the ECM can occur via a multicellular process (collective

invasion, cell streaming) and individually (amoeboid migration, mesenchy-

mal migration). Migration via these routes is dependent on tumor cell type

and the underlying microenvironment [24] (Fig. 6.4).

Collective migration is the movement of whole clusters or sheets of

tumor cells. This occurs when two or more cells retain cell–cell junctions

and move together through the ECM. Leading edge cells participate in che-

motaxis and ECM degradation to create space.

Amoeboidmigration does not always requireMMP activity because cells

can cross the space of the basal membrane by altering their biomechanical

properties. ECM degradation is, however, required for mesenchymal

Figure 6.4 Tumor cell migration. (A) Collective cell invasion and/or cell streaming.(B) Individual cell invasion.


migration. These processes may be coincident and can interconvert in

response to the microenvironment and regulatory factors such as micro-

RNA (miRNA) [25].

Tumor cells face considerable obstacles during invasion. For example,

E-cadherin (CDH1) is a critical element in epithelial tissue organization that

mediates intercellular unions and prevents dissociation of epithelial cells.

As such, tumor cells choose the epithelial–mesenchymal transition (EMT)

as an option for invasion. The BM barrier can be degraded by invadopodia,

specialized protrusions rich in F-actin, present on the tumor cell membrane

[26–28].

1.3. Epithelial–mesenchymal transitionTheEMT is a process inwhich cells lose their epithelial characteristics (cell–cell

adhesion, apical–basal polarity) and acquiremesenchymal properties (invasive-

ness, resistance to apoptosis, motility) [29,30]. Interaction between cancer cells

in the tumormicroenvironment can induceEMTbyauto-andparacrine secre-

tionof growth factors, cytokines andECMproteins. EMTfacilitates tumor cell

migration and invasion of the surrounding microenvironment by weakening

cell–cell cohesion, increasing ECM degradation, and modifying the cellular

cytoskeleton. Epithelial cell cytoskeletal markers include cytokeratins (CKs)

and mesenchymal cells include vimentin (VIM). As can be expected, CK are

usually repressed and VIM overexpressed during EMT [31].

Transcription factors associated with EMT include snail homolog 1 of

drosophila (SNAI1), snail homolog 2 of drosophila (SNAI2), twist homolog

1 of drosophila (TWIST), defensin alpha 1 (DEFA1), zinc finger E-box

binding homeobox 1 (ZEB1), zinc finger E-box binding homeobox 2

(ZEB2), goosecoid homeobox, and forkhead box C2. These factors suppress

epithelial markers and induce the expression of mesenchymal markers.

Signaling pathways include transforming growth factor-beta (TGF-b),NOTCH1, and wingless-type MMTV integration site family (WNT) that

repress the expression of complex cell adhesion molecules, such as CDH1,

claudin, and occludin [32,33].

SNAI1 and ZEB increase the expression of different MMP and plasmin-

ogen system components that promote ECM degradation [34]. SNAI1 and

SNAI2 also participate in neovasculature formation through angiogenic fac-

tors [35]. EMT regulators such as ZEB1 and TWIST enhance intravasation

while manipulating endothelial cell interaction [36,37]. EMT enhances cell

resistance to apoptotic signals, thus contributing to CTC survival in the


bloodstream [38]. SNAI1 negatively regulates caspases, which increases

resistance to cell death [39]. ZEB2 prevents apoptosis of cells by protecting

them from DNA damage [40]. TWIST confers resistance by antagonizing

the proapoptotic effect mediated by v-myc myelocytomatosis viral onco-

gene homolog avian (MYC) [41]. ZEB1 is an important component of neu-

rotrophic tyrosine kinase receptor type 2 (NTRK2)-induced EMT and,

thus, suppresses anoikis [42]. Some miRNA have also been associated with

EMT. For example, cells under EMT have reduced expression of the miR-

200 and miR-205 family, whose principal targets of action are ZEB1 and

ZEB2, specific repressors of CDH1 [43–45]. SNAI1 and TWIST are

involved in chemoresistance and radioresistance in several human cancer

cells [46,47].

The EMT of tumor cells appears transient. EMT markers are not usually

found in distant metastases. It is likely that tumor cells acquire mesenchymal

characteristics to invade stroma, extravasate into the circulation, and once in

the distant organ, regain their epithelial characteristics via mesenchymal–

epithelial transition (MET). As such, it is important to consider EMT and

MET markers in the search for CTC. For example, low CDH1, high

VIM, and expression of N-cadherin (neuronal) (CDH2) (markers of

EMT cells) have been recently identified in CTC [48]. CK negative and

mesenchymal-positive breast cancer patients had a poorer prognosis com-

pared to those without mesenchymal markers [49].

CTC with EMT-type modifications acquire a more aggressive pheno-

type, that is, similar to stem cells. This phenomenon and resistance to con-

ventional therapy may generate more aggressive CTC subpopulations

[30,50].

1.4. InvadopodiaInvadopodia were first described as cell adhesion sites in the form of rosettes,

that is, structures similar to those formed in RSV-transformed chicken

embryo fibroblasts, which degraded the ECM [51]. Formation of

invadopodia is associated with tumor invasiveness and metastatic potential.

Invadopodia can be formed in cell lines obtained from bladder primary

tumors [52,53], colorectal cancer, and squamous cell carcinoma [54], as well

as those invasive in in vitro and animal xenographic models, that is, breast

cancer [55–58].

Invadopodia has been demonstrated in gelatin or fibronectin (2D), ECM

gels of collagen type I, and Matrigel 3D [59], in ex vivo cultures on ECM


peritoneum [54,60] and urinary bladder [61], as well as xenografts by immu-

nofluorescent and intravital imaging. Primary culture invadopodia is mor-

phologically equal to that in cell lines and cortactin positive, a marker of

invadopodia.

Eckert et al. [62] showed that SH and PX domains 2A (SH3PXD2A,

TKS5) were markers of invadopodia. In their absence, breast cancer cells fail

to metastasize to the lungs, a process necessary for hematogenous dissemina-

tion. Gligorijevic et al. [63] showed that altered Wiskott-Aldrich syndrome-

like (WASL orN-WASP) decreased invasion and intravasation, as well as lung

metastasis of mammary tumors. These markers were also associated with

invadopodia.

Elucidation of invadopodia markers is critical to identify cells with meta-

static potential includingCTCand for thedevelopmentofnew therapeutic tar-

gets. These include the IQ motif and Sec7 domain 1 (IQSEC1 or GEP100)

[64], ADP-ribosylation factor 6 (ARF6) [65], cofilin (CFL) [66], epidermal

growth factor receptor (EGFR or mENA) [67], phosphatidylinositol-4,5-

bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA or p110alpha) [68],

cortactin (CTTN) [69], caveolin 1,k caveolae protein 22-kDa (CAV1) [70],

fascin homolog 1, actin-bundling protein (Strongylocentrotus purpuratus)

(FSCN1) [71], MMP-14 (membrane-inserted) [72], FYVE, RhoGEF and

PH domain containing 1 (FGD1) [73], and SH3PXD2A [74]. It is likely that

therapies aimed at disrupting invadopodiawould also injure podosomes, struc-

tures presentona varietyof cell types includingmacrophages, neutrophils, den-

dritic cells, and osteoclasts. Podosomes and invadopodia do, however, possess

differences. Monocytic cell podosomes require growth factor receptor-bound

protein 2 (GRB2) for their formationwhile invadopodia requireNCKadaptor

protein (NCK) [75].

Once tumor cells have degraded the BM, they enter the stroma where

stromal cells enhance neoplastic cells via bidirectional interaction. Adipo-

cytes in the local microenvironment secrete interleukin-6 (IL-6) to promote

tumor cell invasiveness [76], and CD4þ T-lymphocytes stimulate tumor-

associated macrophages (TAMs) to activate signaling mediated by EGFR.

Cancer cells secrete interleukin-4 (IL-4), thereby triggering cathepsin in

TAM and thus increasing invasiveness [15].

1.5. IntravasationNeoplastic cells cross the barrier of endothelial cells and pericytes, which forms

thewalls of the microvessels. The TGF-b [77] and perivascular TAM enhance


the intravasation of breast cancer cells [78] by secretionof epidermal growth fac-

tor and colony stimulating factor 1. Neoplastic cells stimulate the formation of

tortuous new blood vessels with increased permeability. Constant remodeling

facilitates intravasation [79] via several mechanisms involving vascular endo-

thelial growth factor (VEGF), prostaglandin-endoperoxide synthase 2 (prosta-

glandin G/H synthase and cyclo-oxigenase) (PTGS2 or COX-2), epiregulin

(EREG), MMP-1 (interstitial collagenase), and MMP-2 (gelatinase A,

72-kDa type IV collagenase) [80].

1.6. CirculationCancer is a dynamic disease involving time, phenotypic changes, and evo-

lution of neoplastic cells (primary tumor to CTC). As such, CTC represent a

heterogeneous population of cells with unique characteristics that allow dis-

tant travel and the ability to establish disseminated disease.

CTC survive in circulation despite overwhelming obstacles. The latter

include susceptibility to anoikis (epithelial cells that lose their dependent

integrins; adhesion mechanisms normally undergo anoikis) [81], the possi-

bility of being trapped in capillary beds by their relatively large diameter

within minutes of their intravasation, hemodynamic forces, and the innate

immune response (natural killer cells) [14].

CTC apparently modify the pentose phosphate signaling pathway and

the control of glucose uptake [82] and induce NTRK2 expression to suppress

or delay anoikis [83]. They acquire a greater deformation capacity due to a

decreased F-/G-actin ratio resulting in a less polymerized microfilament

network [84]. They evade hemodynamic force and the immune response

by forming relatively large emboli through contact with platelets via expres-

sion of tissue factor and/or L- and P-selectins (SELL and SELP) [85]. This

process may involve microtentacles [86], which are plasma membrane exten-

sions enriched inmicrotubules. The formation of tumor/platelet microemboli

increases the time in which CTC persist until their arrest in distant tissues

[14,86,87]. Despite these evasion strategies, only a small percentage of

CTC ultimately produce secondary tumors.

Specimens obtained from blood and lymphatics are a potential source for

the identification of occult tumor cells. Despite curative treatment, some

patients without evidence of tumor dissemination on clinical, radiological,

pathological, or physical examination will develop recurrent disease. This is

probably due to the presence of scattered occult cancer cells undetected by

routine methods [3].


Metastasis is one of the factors that influence the prognosis and manage-

ment of cancer [88]. The development of high sensitivity immunologic and

molecular methods to detect small numbers of cancer cells in lymph nodes

during primary tumor surgery, in peripheral blood, or distant organs such as

bone marrow, has been attempted [4].

In breast cancer, the presence of metastases in axillary lymph nodes is pre-

dictive of distant metastases. Despite highly sensitivity methods, 20–30% of

patients without axillary lymph node metastases develop distant metastatic

disease. This suggests that hematogenous dissemination is not related to

lymph node metastasis [1].

Detection can be performed by DTC bone marrow aspirate, an invasive

procedure. In comparison, the use of peripheral is a simple and relatively

noninvasive choice that allows repeat sampling. If spread is early, then detec-

tion of tumor cells in peripheral blood is possible before clinical manifesta-

tion in distant organs [3]. According to the metastasis parallel model, the

genetic alterations that CTC acquire should be even more relevant for their

identification and for prediction of the treatment response compared with

the primary tumor.

1.7. Arrest in distant organsCTC spread is generally limited to a target group of organs [89]. It has been

proposed that CTC arrest in passive form due to size constraints of the vascu-

lature in capillaries of distant organs [90]. It has also been proposed that CTC

have predilection for specific tissues. Tumor cells are capable of forming adhe-

sive interactions [91], as well as, sharing and cooperating with the target tissue

via cytokines and chemokines to determine the organotropism of metastasis

[92,93]. Chemokine (C-X-C motif ) receptor 4 (CXCR4) and chemokine

(C-C motif ) receptor (CCR7) and their ligands chemokine (C-X-C motif )

ligand 12 (CXCL12) and chemokine (C-Cmotif ) ligand 21 (CCL21) are used

for breast cancer arrest and permit migration into secondary organs [94,95].

The arrest of metastatic cells at distant sites is facilitated by platelets and

leukocytes that form SELL and SELP complexes with tumor cells. The

expression of selectin ligands (sLex/a, sialyl Lewis x/a glycans) is associated

with metastatic progression and poor prognosis [96].

1.8. ExtravasationCTCmay initiate intraluminal growth and form microcolonies, which even-

tually break the surrounding blood vessel walls to release tumor cells into the


target tissue or organ, or penetrate the layer of endothelial cells and pericytes

by increasing gene expression that promotes vasculature permeability [97–99].

Stroma-derived TGF-b enhances metastatic tropism of breast cancer cells by

lung tissue, inducing angiopoietin-like 4 (ANGPTL4) expression, which, in

turn, facilitates remodeled vasculature and tumor cell extravasation [100].

Other proteins involved in the induction of increased pulmonary vascular per-

meability to allow CTC extravasation are angiopoietin 2 (ANGPT2),

MMP-3 (stromelysin 1, progelatinase), MMP-10 (stromelysin-2), and VEGF

[101–103].

1.9. ColonizationOnce in the target tissue or organ, tumor cells establish a two-way relation-

ship with the microenvironment. They suppress the immune response, pro-

mote angiogenesis, and release factors that promote growth, survival and cell

motility [29]. In response, the host tissue alters gene expression to provide a

favorable environment for tumor cells [104,105]. First proposed by Stephen

Paget in 1889 [106] as the “seed and soil” hypothesis, Paget thought that

successful colonization in a secondary organ depends on the intrinsic prop-

erties of the tumor itself (seed) and on a permissive and supportive role of the

environment (soil).

Each type of cancer (lung, breast, prostate) can metastasize to the same

organ (brain), to develop different molecular programs and activate different

signaling pathways. These are probably related to the accumulation of

genetic changes in cancer cells necessary for primary tumor growth and dis-

semination, as well as, additional changes required to survive and grow

remotely. The genes involved in metastasis are categorized as initiation, pro-

gression and virulence genes, the latter allowing colonization of secondary

organs [96,105,107].

1.10. Metastatic dormancyTumor cells may remain silent and viable for long periods of time, a phe-

nomenon defined as “metastatic dormancy.” This phenomenon has been

implicated in the identification of metastases that are clinically apparent a

few years or decades following surgical resection of the primary tumor.

Tumor cells remain viable and progressively acquire genetic and epigenetic

changes that allow a reduced proliferative and metabolic state thus impeding

therapeutic eradication specifically directed at proliferative and metaboli-

cally active cells [96,108,109].


2. TECHNIQUES FOR CTC DETECTION

CTC detection is a proverbial “needle in the haystack” problem.

Human blood contains an overwhelming number of red cells (5–9�109

per mL), white cells (5–10�106 per mL), and platelets (2.5–4�108 per

mL) relative to CTC (�1 CTC per 106–107 mononuclear cells, MNCs).

Due to their scarcity and high cellular background, these rare events need

to be detected using enrichment techniques combined with ultra-high sen-

sitivity methods [110,111].

Although enrichment techniques increase rate of isolation, there is inev-

itable CTC loss [112] despite the use of markers that uniquely identify these

cells [3]. Tumor-specific markers are key to CTC detection. Epithelial cell

and specific cancer markers are currently in use.

Epithelial markers are usually expressed by all tumor cells of epithelial

origin including epithelial cell adhesion molecule (EPCAM) and several

CKs such as CK7, CK8, CK18, and CK19.

Specific tumor markers used for breast cancer CTC include v-erb-b2

erythroblastic leukemia viral oncogene homolog 2, neuro-/glioblastoma-

derived oncogene homolog avian (ERBB2 or HER-2/neu), secretoglobin

family 2A, member 2 or mammaglobin 1 (SCGB2A2), melanoma antigen

family A3 (MAGEA3), mucin 1, cell surface associated (MUC1), mucin 2,

oligomeric mucus/gel-forming (MUC2), N-acetylgalactosaminyltransferase

(GALNT), serpin peptidase inhibitor, clade B (Ovalbumin), member 5 or

maspin (SERPINB5), and stanniocalcin-1 (STC1).

miRNA are small noncoding RNA molecules that modulate expression

of target genes. These have been reported as stable blood-based biomarkers

in carcinomas. Epigenetic alterations which are common and specific to

tumors have also been proposed as other potential CTCmarkers [113–115].

Theuseof specific tumormarkers detected by immunological ormolecular

techniques, ie, reverse transcription polymerase chain reaction (RT-PCR)

has expanded rapidly. Due to tumor heterogeneity, there is no universally

expressed marker in any particular cancer type.

On the other hand, “tumor-specific” markers are also expressed by nor-

mal cells such as leukocytes, albeit at lower levels. For this reason, genetic

disorders or abnormalities found exclusively in tumor cells, such as ERBB2

amplification in breast cancer, have been proposed as tumor specific. PCR

or fluorescence in situ hybridization (FISH) can detect these genetic

alterations.


These issues have resulted in the lack of acceptance as a standard method

in the diagnosis, management, and monitoring of cancer [116].

2.1. Enrichment or isolation methods2.1.1 Cell morphology2.1.1.1 Epithelial tumor cell isolationEpithelial tumor cell isolation (ISET) is based on differences in the cancer

cell size versus peripheral blood leukocytes. Tumor cells are usually larger

than their normal cell counterparts and may be isolated by microfiltration

methods [117,118].

ISET uses a microfluidic platform consisting of multiple arrays of isola-

tion wells of increasing size. Wells (5–8 mm) permit passage of leukocytes

and deformable erythrocytes while entrapping large tumor epithelial cells.

This approach requires 6–15 mL peripheral blood and has an isolation effi-

ciency and cell purity of 80% for breast and colon cancer cells with a sen-

sitivity of one CTC per milliliter. CTC are isolated without damaging

inherent morphology.

CTC are evaluated by cytopathologists for malignant phenotype by

morphologic criteria including nuclei equal to or greater than two pores

(16 mm), nuclear contour irregularity, visible presence of cytoplasm, and a

high nucleus/cytoplasm index (>0.8).

Cell identification and characterization can also be performed by immu-

nocytologic, cytogenetic, and molecular studies. Cells of interest can then be

individually recovered by laser microdissection and their DNA amplified.

The limitations include low specificity, potential to lose smaller CTC,

and retention of microemboli and lymphocytes larger than the pore size

[118–120].

2.1.1.2 ScreenCell isolationScreenCell is a system designed for the collection of peripheral blood CTC

in using small compact devices containing a microporous membrane that

allows size-selective isolation of rare tumor cells. Following lysis of red blood

cells (RBC), CTC isolation is achieved in 3 min in standard collection tubes.

The circular track-etched filter (polycarbonate, it4ip, Belgium) is 18 mmthick, with a smooth flat and hydrophilic surface. Circular pores are cali-

brated (7.5�0.36 or 6.5�0.33 mm for isolation of fixed or live cells, respec-

tively) and randomly distributed throughout the filter (1�105 pores/cm2).

This system can isolate and characterize a wide variety of tumor cells, both

live and fixed, including mesenchymal, cancer stem, microemboli, and


cancer cells of nonepithelial origin. Cells are well preserved and can be iso-

lated and grown in culture for molecular studies or screening potential

therapeutics [121].

2.1.2 Enrichment based on cell density2.1.2.1 Ficoll-HypaqueThis density-based method is based on differential centrifugation in Ficoll-

Hypaque (GE Healthcare). The density of MNCs is<1.077 g/mL, whereas

that of other blood cells and granulocytes is higher [112]. Separation of cell

types is accomplished in different layers or strata [122] (Fig. 6.5). This

method has a sensitivity of one CTC per 4.6 mL and requires 15–30 mL

blood. Isolated CTC can be identified and characterized by immuno-

cytologic, molecular, and cytogenetic analyses. This approach is relatively

easy to perform and inexpensive. However, it has low specificity and

involves cross-contamination with polymorphonuclear cells. In addition,

CTC may be easily lost. Centrifugation must be performed immediately

to prevent mixing between the layers. Ficoll-Hypaque may be toxic to cells

in prolonged contact thus changing gene expression [112].

2.1.2.2 OncoQuickThis method increases the efficiency of density gradient centrifugation

(OncoQuick, Greiner Bio-One, Germany). To avoid cross-contamination,

a porous membrane is used to separate reticulocytes and granulocytes

from tumor cells and MNCs. The average tumor cell recovery rate is

improved (87%) versus traditional Ficoll-Hypaque (84%). This method

Figure 6.5 Density gradient separation. Mononuclear cells (MNCs) containing CTC aresuspended and erythrocytes pelleted.


reduces MNC contamination, thereby improving CTC characterization by

RT-PCR [122].

2.1.3 Immunomagnetic enrichmentImmunomagnetic enrichment is based on the differential expression of spe-

cific surface antigens on epithelial tumor cells and mononuclear hematopoi-

etic cells using both positive and negative selection. In the positive pathway,

CTC are selected via magnetic beads coupled to antibodies directed to epi-

thelial surface markers, such as CK, EPCAM, or tumor-specific antigens,

that is, carcinoembryonic antigen or ERBB2. The most commonly used

is EPCAM, a cell adhesion molecule expressed on the surface of 80% of can-

cers of epithelial origin [123,124]. False positive selection may occur due to

the expression of epithelial markers in nonepithelial cells [3,125] (Fig. 6.6).

In negative selection, reduction is carried out using MNC surface

markers of hematopoietic cells [126,127]. The most commonly used are

CD45 and CD60, surface markers expressed only on leukocytes and mega-

karyocytes, respectively [128].

2.1.3.1 Magnetic activated cell sortingMagnetic activated cell sorting (MACS) captures CTC by immunolabeling

superparamagnetic particles (�50 nm diameter) (Miltenyi Biotec, Germany).

These beads are composed of a biodegradable matrix. It is therefore not

Figure 6.6 Immunomagnetic cell isolation.


necessary to remove them from cells after the separation process. Isolated cells

are preserved in structure, function and activity.

This approach can result in false positive results due to nonspecific label-

ing or false negatives due to the absence of CTC antigens. MACS has a sen-

sitivity of one cell per 0.3 mL requiring 5–15 mL blood [129,130]. CTC

isolated by this method can be characterized by immunocytologic, molec-

ular, and cytogenetic studies.

2.1.3.2 DynabeadsDynabeads (Dynak, Norway) are uniform spherical polystyrene beads that

have been made magnetizable and superparamagnetic, that is, magnetic only

in a magnetic field. The beads can easily be resuspended when the magnetic

field is removed. The target-specific antibodies bind to the surface of the

beads, allowing the capture and isolation of intact CTC. The antibodies,

or ligands, may be covalently bound to the magnetic beads or bound via sec-

ondary antibodies. CTC may be removed by specific antibodies, enzymatic

cleavage or reaction with affinity molecules.

Dynabeads can be used for isolation or depletion of various cell types

using positive selection or negative selection or both. Positive selection

results in a bead:cells ratio of 4:1–10 with high purity (95–100%) and via-

bility (60–95%) using >1�107 beads/mL. Negative selection results in a

bead:cell ratio of >4:1 with high depletion (95–99%) using >2�107

beads/mL. Successful cell isolation with this approach is dependent on mag-

netic bead concentration, bead/target cell index, and antibody [131].

2.1.3.3 EasySepEasySep is an immunomagnetic selection method (Stem Cell Technology,

Vancouver, Canada). The cells are labeled with monoclonal antibodies

directed against specific cell surface markers. The method employs tetra-

meric antibody complexes (TACs) and dextran-coated magnetic particles

to select or deplete cells of interest. Magnetic separation results in two dis-

tinct cell fractions that are immediately available for analysis [132].

2.1.3.4 RosetteSepRosetteSep is an immunogenicity gradient method based on negative selec-

tion (Stem Cell Technology, Vancouver, Canada) [133]. This method uses

autologous RBC already present in the sample as dense particles to pellet

unwanted white cells, thereby purifying specific cell subsets by negative

selection. A cocktail of TACs that target multiple cell types crosslink


unwanted cells to many RBC in whole blood. Bispecific TACs bind RBC

to the target cells and monospecific anti-RBC x anti-RBC TAC bind addi-

tional RBC, thus forming a “rosette.” The sample is then layered on a buoy-

ant density medium and centrifuged. The rosettes pellet, along with any free

RBC and granulocytes. CTC, which have not been labeled with TAC and

are not linked to RBC, do not pellet and are recovered at the plasma

interface [132].

2.1.3.5 Flow-based immunomagnetic detectionIn 2007, Immunicon presented an automated flow-based immunomagnetic

detection system in which the selection of epithelial cells is accomplished by

antibody-coated ferrous particles that separate EPCAM-expressing epithe-

lial cells. Cells are labeled with monoclonal antibodies to CK, CD45 (a pan-

leukocyte marker), and 40,6-diamidino-2-phenylindole (DAPI, a nuclear

stain) and characterized by fluorescent microscopy. The CK and DAPI pos-

itive cells are counted as a positive event and expressed as CTC per milliliter.

CK cells and/or DAPI negatives, as well as CD45 positives, are counted as

negative events. Unfortunately, CTC lacking antigen expression are not

detected [3].

2.1.3.6 CellSearchCellSearch is a semi-automated immunomagnetic separation system

(Veridex) approved by the FDA for detection and analysis of CTC in met-

astatic breast, prostate, and colorectal cancer. This method uses ferrofluid

nanoparticles labeled with EPCAM antibody. Following a three-step label-

ing procedure to distinguish CTC from leukocytes (CK8, CK18 and CK19,

DAPI, CD45), CTC are retained by application of a magnetic field

[134–136].

CTC are defined as intact cells with oval morphology of at least 4 mm in

size, positive for DAPI (nuclei) and CK (bright or moderate), and negative

for CD45/APC (leukocyte cytoplasm). Two cut-off points for whole blood

positivity have been defined: two or more CTC [134] and five or more

CTC per 7.5 mL [135].

This system requires a moderate amount of blood, is commercially avail-

able and has good recovery rate (82%). Sensitivity is one cell per 0.5 mLwith

high reproducibility and specificity [130]. This method, however, selects

only EPCAM positive CTC. False positives due to labeling of nontumor

cells and false negatives from loss of EPCAM can also occur.


2.1.4 Microfabricated devices2.1.4.1 Affinity-based chip (CTC-chip)This system uses a microfluidic chip composed of 78,000 microposts

(100 mm height and 100 mm diameter) fabricated by deep reactive ion etch-

ing in silicon (total post surface area 970 mm2). The gap between posts

(50 mm) limits the flow rate to allow cell interaction with the microposts

functionalized with EPCAM antibodies for CTC capture.

The device has 60–65% capture efficiency (flow rate <2 mL/h) and

uses 0.9–5.0 mL blood. Real-time monitoring [137] and allows for the

isolation of highly viable cells (98%). Despite high sensitivity and specific-

ity, only EPCAM positive CTC are selected. Loss of EPCAM in CTC

leads to false negative and nonspecific binding leads to false positive results

[3,137,138].

2.1.4.2 Microvortex-generating herringbone-chipThis version of the CTC-chip uses surface ridges, that is, herringbones, in

the wall of the device to disrupt the laminar flow streamline to maximize

collisions between CTC and the EPCAM antibody-coated walls [139]. This

device isolates CTC directly from blood thus preventing potential cell loss

and destruction. Highly viable cells are obtained.

2.1.4.3 Deterministic flowThe deterministic hydrodynamic flow microfluidic device is based on

particle size separation. The microfluidic chamber consists of a micropost

array. Micropost diameter, distance between microposts in each row and

row-to-row variation are important design parameters. Particles below

and above the critical hydrodynamic diameter follow different trajectories,

that is, separation.

This device has been used for the separation of various blood cell

types and DNA fragments. Device flow rates (1 mL/min) correspond to

a flow velocity of 1 mm/s, approximate to that physiologically [3,138,140].

2.1.4.4 DEP chipThe DEP (dielectrophoresis) chip uses thin flat chambers with microelec-

trode arrays patterned by microfabrication. The DEP force arises when

AC fields are applied to the electrodes. The nDEP (negative dielectrophoresis)

force and the hydrodynamic force vary at different distances from the elec-

trodes. Cells with similar properties find their steady-state positions between

electrodes. Since the flow profile of a cross section along the flow direction is


almost a perfect parabola, particles in fluid layers at different distances have

different flow velocities. Cells of different types are separated with respect

to their dielectric properties and hydrodynamic flow properties without

impacting viability [3,138,141–143].

DEP chip has an efficiency of 100% when MDA-MB-231 breast cancer

tumor cells are spiked in blood at tumor:blood cell ratio of 1:105 with a rate

of separation of 103 cells/s [141]. This method is limited by its sample vol-

ume thereby reducing its ability to identify small number CTC. This

approach also requires separation of the cellular components from plasma

and resuspension in a low conductivity isotonic sucrose and dextrose

medium.

2.1.4.5 Size-based microfluidic separationThis filter method is based on size because most CTC are larger than hema-

topoietic cells. The filter separates CTC in a simple rapid manner (10 min)

and does not require use of antibodies. A recovery rate of>85% for cultured

prostate cancer cells spiked in blood has been reported [3,138,144].

2.1.5 Other cell enrichment systems2.1.5.1 AdnaTest BreastCancerAdnaTest (AdnaGen AG, Langenhagen, Germany) isolates EPCAM posi-

tive cells via magnetic beads conjugated with epithelial cell antibodies

[145]. CTC are lysed and mRNA examined by RT-PCR to identify

tumor-specific markers (MUC1 and ERBB2). This method has a sensitivity

of two cells per 5 mL and takes into account tumor cell heterogeneity in

gene expression patterns. The test is considered positive if at least one or

more of three markers show increased signal expression (�0.15 ng/mL).Like other methods, there is the possibility of false positives by nonspecific

labeling and of false negatives by antigen loss [136,145].

Using this method, Andreopoulou et al. [146] demonstrated that ERBB2

amplification in the primary tumor resulted in expression of ERBB2 in CTC.

Other studies, however, reported discordance in ERBB2. In metastatic

breast cancer, ERBB2-positive CTC were found in 41% and 47% of cases

using CellSearch and AdnaTest BreastCancer, respectively [147]. Using Cel-

lSearch, ERBB2-positive CTC were detected in 32% of patients with

ERBB2-negative primary tumors. Using AdnaTest BreastCancer, 49% were

detected. There was no concordance in the ERBB2 positivity between the

primary tumor and CTC (p¼0.96, k¼�0.006). In the GeparQuattro trial,

ERBB2 overexpressing CTC were observed in 14 of 58 patients with breast


cancer (24.1%). Eight of the patients (57%) had ERBB2-negative primary

tumors [148].

Apparently, there is a subgroup of patients with an ERBB2-negative pri-

mary tumor who develop CTC with ERBB2 amplification. Assessment of

this expression in CTC may help identify patients who initially would not

have been considered candidates for targeted therapy against ERBB2

[149,150].

2.1.5.2 Collagen matrix adhesion assay (CAM)This cellular enrichment system is based on invadopodia and the tendency of

tumor cells to invade collagen matrices. This approach is accomplished by

positive (EPCAM, pan-CK, and recapture of CAM fragments) and negative

selection (CD45). The CAM-coated device successfully recovered tumor

cells spiked in 1 mL blood with a 54�9% recovery rate and 0.5–35% purity,

and detected invasive tumor cells in 100% of patients with metastatic breast

cancer (18–256 CTC per mL). It has good sensitivity (one cell per 0.1 mL)

and excellent cell viability (99%) for subsequent molecular and genetic anal-

ysis. Although requiring only 3 mL blood, samples must be cultured for

12 h [151].

2.1.5.3 CTC infectionInfection with green fluorescent protein (GFP) expressing adenovirus, that

is, fluorescent protein gene [152] has been used to detect small numbers of

viable CTC [153]. This three-step detection method involves lysis of RBC,

addition of telomerase-specific replication-selective adenovirus expressing

the GFP (OBP-401, TelomeScan), and fluorescent microscopy.

This method allows precise enumeration of human CTC because OBP-

401 can replicate and fluoresce only in viable tumor cells [152]. The signal is

typically detected in the incubated cells 4–12 h postinfection. Because this

virus causes human infection, its usefulness may be limited.

2.1.5.3.1 NV1066 NV1020 is a genetically engineered cancer-specific

herpes simplex virus. This virus has been further modified by insertion of a

gene for enhanced GFP into the genome, creating NV1066. Enrichment is

based on infection of cancer cells with NV1066 via expression of GFP and

molecular analysis.

This fast and simple method has a sensitivity of 100 CTC per 5�107

blood cells and requires no molecular training. However, false positives

can be result from NV1066 phagocytosis by activated immune cells.


CTC antigens may occur after a long period of incubation with

NV1066 [153].

2.1.5.4 Electrical biosensorThis semi-integrated electrical biosensor detects rare CTC in blood. The

sample is enriched by immunomagnetic isolation (anti-EPCAM coated

beads) and size filtration. When these methods are combined, recovery rates

are >70%, even at low CTC. The enriched sample is then transferred to a

microchip for magnetic concentration, followed by immunochemical trap-

ping and electronic detection by impedance spectroscopy [154].

2.1.5.5 Fluid biopsyFluid biopsy is a relatively simple process with minimal processing of blood

samples and subsequent pathologic or cytopathologic examination.

Marrinucci et al. [155] reported the identification of CTC without using

protein-based enrichment markers. Following erythrocyte lysis, nucleated

cells are pelleted and resuspended in with phosphate buffered saline

(PBS). Cells are applied to glass slides using 2% paraformaldehyde and cold

methanol used for permeabilization. Nonspecific binding sites were blocked

with goat serum, and the slides were incubated with monoclonal anti-

pan-CK antibody (Sigma) and CD45-Alexa 647 (Serotec). Then they were

washed (PBS) and incubated with Alexa Fluor 555 goat antimouse antibody

(Invitrogen). The cells were DAPI counterstained.

Slides (four per patient) were scanned using a fast scanning fluorescent

microscope. Images were subjected to an analysis algorithm to identify high

definition-CTC (HD-CTC) based on CK and CD45 intensity, shape, and

nuclear and cytoplasmic size, as well as intact DAPI nucleus without apo-

ptosis. Background WBC provides morphologic comparison.

Using this technique, CTC were identified in the majority of metastatic

cancer cases. This method allows for subsequent HD-CTC characterization.

Samples can be stored frozen for an extended time for additional studies.

Evidence suggests that CTC obtained by this method have similar morphol-

ogy to those cells found in the primary tumor [155].

2.1.5.6 iCeapIntact CTC enumeration and analysis procedure (iCeap) uses immuno-

magnetic enrichment and flow cytometric analysis for contamination-free

measurements [156]. The microfluidic chip allows collection of CTC for

subsequent RT-PCR, chromosome aneuploidy and mutation analysis.


Enrichment is performed with MACS EPCAM-MicroBeads and cells

are labeled with an allophycocyanin (APC) conjugated EPCAM monoclo-

nal antibody. This method produces intact high viability CTC.

2.2. CTC identificationFollowing enrichment, identification of CTC is necessary to assess origin.

Identification methods may or may not be separate from the enrichment

procedure and are usually protein-or nucleic acid-based [157–166].

2.2.1 CytometryCytometric methods can isolate and enumerate cells based on their antigen

expression via monoclonal antibodies directed against epithelium- or CTC-

specific markers. This approach allows for quantification of cell subpopula-

tions and simultaneous examination of cell size, viability, and DNA content,

as well as intra and extracellular markers [156,157]. CTC obtained by this

method may undergo additional morphologic assessment and molecular

analysis.

This approach frequently relies on the detection of cell surface CK.

Unfortunately, CK may be down regulated in tumor cells to allow invasion

and migration [158,159]. Although ERBB2 is another potential marker for

CTC [160], it is not present in all tumor cells and is expressed in 10% of

healthy women [161]. EPCAM is expressed in most epithelial tumors and

can be used as a CTC marker [162,163]. It has low sensitivity (one tumor

cell/104–105 blood cells) compared to RT-PCR [167,168].

2.2.1.1 FASTFiber-optic array scanning technology (FAST) (Bruce Laboratory, Palo Alto

Research Center) is a fiber-optic array laser-scanning technology. This

method enables rapid high-fidelity location of CTC identified by the con-

ventional markers CK, DAPI, and CD45. CTC can be simultaneously

labeled for at least three additional tumor biomarkers.

FAST locates CTC 500-fold faster than automated digital microscopy

(ADM) with comparable sensitivity, but improved specificity. FAST does

not require cell enrichment. FAST has a light collection system that has a

very large field of view (50 mm) with no loss of efficiency. This wide col-

lection aperture (100-fold increase over ADM) is large enough to enable

continuous scanning and eliminates the need to step the sample, that is,

the main source of latency [169]. A database containing true and false pos-

itives allows optimization of image filters [169–171].


2.2.1.2 EPISPOTThe epithelial immunospot technique (EPISPOT) detects proteins

secreted/released/shed from single epithelial cancer cells. Cells are cultured

on an antibody-coated membrane. Following capture, fluorochrome-

labeled secondary antibodies are used subsequently detect proteins such as

CK19 and MUC1 in breast cancer.

Only viable cells are identified, as nonviable cells are unable to secrete

adequate amounts of protein. EPISPOT has a 100-fold higher sensitivity

in detecting CK19 in breast cancer cells versus ELISA [172,173].

2.2.1.3 LSCLaser scanning cytometer (LSC) (Compucyte Corporation, Cambridge,

MA, USA) combines the speed of flow cytometry with the ability to analyze

every positive event morphologically.

Combined with antibody-coated magnetic beads (EPCAM and CD45),

the cytometer determines background fluorescence dynamically to calculate

peak and integral fluorescence on a per-cell basis. This calculation results in

improved correction for background fluorescence. It is possible to relocate

the cells within the positive population allowing microscopic verification.

One positive cell in 104 cells is detectable, and 50 out of 60 tumor cells

are reliably recovered from 20 mL blood (�1–2 cells per 107 blood cells)

after magnetic bead enrichment [112,157,174,175].

2.2.1.4 Automated microscopic systemThe automatedmicroscopic system detects and quantifies CTC. It also stores

each detected image for subsequent visual assessment.

Kraef et al. [176] developed the rare event imaging system for the detec-

tion and analysis of cancer cells in blood and bone marrow. Slides are auto-

matically scanned at low magnification for tumor cells (CK/rhodamine

labeling) and total cell count (nuclear DAPI labeling). All positive events

can be reviewed and confirmed with higher magnification. Cells may also

be viewed using fluorescence filters for multiple-marker analysis to increase

specificity and phenotypic characterization. Kraef et al. [176] developed a

double-labeling protocol that combined CK with breast cancer surface anti-

gens, that is, EPCAM [177,178].

Other automated scanning systems are available including ACIS

(Automated Cellular Imaging System, DAKO, Glostrup, Denmark) and

ARIOL (Applied Imaging Corp., San Jose, CA, USA) [157].


2.2.2 Methods based on nucleic acidsBreast cancer CTC may be identified by detection of genetic or epigenetic

alterations including mutations in proto-oncogenes or tumor suppressor

genes, microsatellite instability and presence of oncogenic viruses [157].

Because circulating free DNA may not be reflective of actual tumor cell

presence, this method has not been clinically implemented.

2.2.2.1 RT-PCRSeveral RT-PCR methods for analysis of epithelium- or organ-specific

expression may facilitate investigation of target genes relevant to metastasis

[179]. Detection of mRNAof overexpressed or mutated genes in breast can-

cer by RT-PCR has been considered a suitable alternative in CTC detec-

tion. As RNA disappears quickly from the blood after cell death, detection

of RNA is likely due to the presence of whole tumor cell, not cell fragments

or free RNA. Following cDNA synthesis, the gene of interest is amplified

using traditional oligonucleotide primers [157].

RT-PCR has high sensitivity for CTC detection in peripheral blood

[180]. This rapid method requires a small blood volume (1.5–10 mL) and

has a sensitivity of approximately one tumor cell/106 blood cells. Due to

its high instability, loss of RNA can lead to false negative results.

False positives can be detected using tumor markers of low specificity to

detect nonmalignant epithelial cells released during an invasive procedure,

for amplification of free nucleic acids in peripheral blood or markers with

illegitimate expression in nontumor cells (hematopoietic cells) from sample

contamination [181] or from the presence of pseudogenes. False negatives

may occur due to the absence or decreased expression of marker genes

[66,88,182,183]. Unfortunately, this technique does not allow CTC visual-

ization or subsequent analysis by other methods. It cannot distinguish viable

and nonviable cells [151].

Markers such as SERPINB5, SCGB2A2, and CK19 permit the use of

this technique to detect CTC [182,184,185]. Multiplex RT-PCR permits

detection based on the expression of various tumor-associated genes.

2.2.2.2 qRT-PCRqRT-PCR allows visualization of low and high mRNA expression thus

increasing discrimination of normal versus tumor cells. Unfortunately, the

presence of a specific marker in breast cancer is inconsistent due to tumor

heterogeneity.


The use of internal qRT-PCR controls minimizes false positives and

improves specificity of CTC detection. Repetitive measurement signal

amplification also allows the identification and elimination of false positive

results [186]. This approach can detect specific CTC transcript fragments in

the blood of cancer patients even when CTC are not viable and have not

been detected by other methods [117].

The selection of appropriate mRNA markers specifically expressed in

tumor cells is critical for achieving the highest CTC sensitivity and specific-

ity. The sensitivity of the SCGB2A2 marker by qRT-PCR is 29–49% in

patients with metastatic breast cancer [130,187]. The sensitivity of bac-

uloviral IAP repeat containing survivin (BIRC5) and telomerase reverse

transcriptase (TERT) in the peripheral blood of patients with breast cancer

is 36.2% and 59.6%, respectively [188]. Sensitivity of CTC detection

increased to 70.2% when BIRC5, SCGB2A2, and TERT were combined.

Using qRT-PCR, MUC1 expression was associated with 11%, 24%,

and 45% of patients with benign breast disease, breast cancer, and operable

advanced breast cancer, respectively [128,189]. Secretoglobin family 2A,

member 1 or mammaglobin 2 (SCGB2A1) was found to be one of the best

CTC markers in breast cancer.

3. MOLECULAR AND GENETIC CHARACTERIZATION

Some patients do not develop metastases even in the presence of CTC

in the peripheral blood [190,191]. As such, the characterization of these cells is

vitally important and may be accomplished by a variety of techniques includ-

ing immunohistochemistry (IHC), FISH, comparative genomic hybridization

(CGH), PCR, RT-PCR, gene expression microarrays, and others.

These methods provide important information regarding malignancy

and metastatic potential and whether or not CTC are genetically identical

to the primary tumor [130,147,149].

3.1. IHCIHC can detect tissue-specific antigens and markers of invasiveness, angio-

genesis, apoptosis, and cell proliferation, among others.

3.2. FISHFISH can be used to identify chromosomal abnormalities via labeled probes

that target-specific DNA sequences. More than one probe may be used at

the same time, that is, each probe labeled with a different fluorochrome.


The most useful FISH probes are: centromeric, chromosome painting, and

locus-specific for fusion, deletion or duplication studies.

Centromeric enumeration probes hybridize to the alpha (or beta) satellite

repeat sequences within the centromeric region specific to each chromo-

some. As such, these probes are used for chromosome enumeration, that

is, detection of ploidy abnormalities. Chromosome painting probes, gener-

ated from chromosome-specific probe libraries, are useful in deciphering

cytogenetic aberrations. Locus-specific probes hybridize to a unique human

genome sequences. They are most frequently used to detect rearrangements,

gains, and deletions as well as amplification in bothmetaphase and interphase

cells. FISH interphase analysis is an attractive and practical way to assess

ERBB2 amplification [192].

Evaluation of the tumor and its CTC by FISH has revealed that a neg-

ative ERBB2 primary lesion can release positive ERBB2 CTC and vice-

versa [149].

3.3. Multicolor FISHMulticolor FISH is based on the simultaneous hybridization of 24 chromo-

some-specific probes. This approach is suitable for identification of cryptic

chromosomal aberrations, that is, translocation of telomeres, marker chro-

mosomes, and unbalanced chromosomal translocations. Unique color pat-

terns are produced by labeling each chromosomewith a single fluorochrome

or with combinations of multiple fluorochromes [192].

3.4. CGHCGH is a quantitative two-color FISH technique [193]. CGH detects geno-

mic imbalances in solid tumors or any desired genome and determines the

map position of chromosomal gains and losses, or chromosomal sub-regions,

on normal reference metaphase preparations. CTC DNA (labeled green)

and normal reference DNA (labeled red) are competitively hybridized to

normal human metaphase spread. The reference DNA serves as a control

for local variations in the ability to hybridize target chromosomes. Digital

image analysis provides a ratio of green-to-red fluorescence along the chro-

mosome on the reference metaphase spread, reflecting the copy number of

the corresponding sequences in the CTC DNA [192].

This technique can detect changes in copy number in DNA without

knowledge of suspicious genetic aberrations. It has been used to observe

genomic instability and the heterogeneity of CTC and DTC [194]. Because

its resolution is 2–4 mb, some alterations below this level cannot be excluded


in those EPCAM and CK19 positive CTC that do not possess CGH changes

[194]. Ploidy aberrations escape detection by this technique [192].

3.5. Array CGHThis methodology has a resolution of 100 kb to 1 Mb. DNA arrays consist of

2000–4000 BAC clones representing the sequenced genome. Oligonucle-

otide arrays are also used in copy-number detection. These arrays contain

25-mer oligonucleotides originally designed to assess human single-

nucleotide polymorphisms. This method measures allelic loss of heterozy-

gosity [192]. Array CGH has revealed a copy number variation in which

thousands of regions of the human genome are now known to vary

[195]. This methodology is a powerful strategy to identify genes and assess

their oncogenic capacity in breast cancer cells including CTC [196].

3.6. Multimarker quantitative real-time RT-PCRNakagawa et al. [197] developed a qRT-PCR multimarker (STC1,

GALNT, and MAGEA3) approach to measure CTC in early stage breast

cancer. At least one biomarker was detected in 29%, 45%, and 77% in stage

I, II, and III cancer, respectively.

3.7. AmpliGridAmpliGrid is a PCR-based chip for direct analysis of individual cells to iden-

tify differences between CTC (Beckman Coulter Genomics, Munich, Ger-

many). In this system, 48 single cells are deposited in separate positions on a

slide and analyzed individually. Using single cell PCR, deposited cells can be

“typed” using epithelial antigens, CK, specific tumor genes, and miRNA.

Cells may be screened quantitatively for diagnostic and/or prognostic

RNA and DNA markers. The ratio of CTC to normal cells can be rapidly

determined in native samples of peripheral blood [112].

3.8. RNA in situ hybridizationIn situ hybridization (ISH) is a method that identifies CTC tumor-specific

mRNA in situ. ISH can simultaneously detect multiple transcripts using pro-

bes for different genes that may have the same recognition sequence for signal

amplification, thereby generating a “pooled” signal. Alternatively, multiple

and independent amplification signals may be used to simultaneously detect

species of target RNA with different signals [163,198]. Two ISH-based sys-

tems have been developed.


3.8.1 CTCscopeTheCTCscope (Advanced Cell Diagnostics, Inc., Hayward, CA, USA) uses

a panel of mRNA epithelial and tumor markers to detect individual viable

CTC in patients with metastatic breast cancer [199]. Following enrichment,

ISH is performed using a panCTC marker panel (CK5, CK6, CK8, CK14,

CK17, CK18, CK19, CK20, EPCAM, MUC1, VIM, TWIST, CDH2,

fibronectin 1 (FN1), and CD45). Data are evaluated using a computer algo-

rithm described by Bushnell et al. [200].

A sample is rated for CTC scoring if PBMC are stained positively with

CD45 indicating acceptable RNA integrity. CTC are identified by a strong

positive panCTC mRNA staining. CTC images are acquired with the

nuance multispectral imaging system (CRI, Cambridge, MA, USA), using

a combination of long-pass filters. Overlapping signals are separated by com-

parison to a reference library.

Using this approach, a trial was conducted in 45 patients with metastatic

breast cancer [199]. When compared to CellSearch, both techniques had

similar CTC sensitivity. CTCscope detected CTC in 47% of patients,

whereas CellSearch detected CTC in 51% of patients. Overall agreement

was 69% (33% positive and 36% negative).

3.8.2 RNAscopeRNAscope (Advanced Cell Diagnostics, Inc.) uses novel RNA ISH technol-

ogy with a probe design strategy that allows simultaneous signal amplification

and background suppression to achieve single-molecule visualization while

preserving cell morphology. RNAscope provides the opportunity to profile

single cell gene expression in situ, unlocking the full potential of RNA bio-

markers. The targeted molecular signature of every cell in a sample is revealed

and precisely measured.

The assay consists of target probes and a signal amplification system com-

posed of preamplifiers, amplifiers, and label probes. Following fixation, cells

are permeabilized using protease to allow probe access. The target probes

are standard oligonucleotides that are designed to hybridize as pairs, with each

pair creating a binding site for a preamplifier. The preamplifier is hybridized to

the target probes at a temperature that favors hybridization to target probe

pairs, but not individual target probes. This ensures that if unpaired target pro-

bes hybridize nonspecifically to a nonspecific RNA, no signal amplification

will occur. The amplifier is then hybridized to the preamplifier, and the label

probe, which is conjugated to either a chromogenic or fluorescent molecule,

is hybridized to the amplifier. The stained samples can then be visualized

under a standard bright field or fluorescent microscope [163].


Each set of target probes spans a region of approximately 1 kb of the tar-

get RNA and hybridizes to 29 preamplifiers. Each preamplifier can hybrid-

ize to 20 amplifiers and each amplifier can hybridize to 20 label probes. This

results in over 8000 fluorescent molecules spanning just 1 kb of RNA [163].

3.8.3 Quantigene ViewRNAQuantigene ViewRNA system is an RNA ISH assay (Affymetrix, Fremont,

CA, USA). It enables multiplex, single-molecule RNA sensitivity and spec-

ificity by using simultaneous branched DNA (bDNA) signal amplification

and background suppression.

In combinationwith ScreenCell isolation, this approach integrates rare cell

isolation and molecular characterization [121]. Peripheral blood is processed

using a ScreenCell isolation device. CTC are transferred to a 24-well cell cap-

ture plate for enumeration/characterization using QuantiGene ViewRNA

ISHCell Assay. A target-specific probe set containing 20 oligonucleotide pairs

hybridizes to the target RNA. An oligo pair hybridization event is essential for

signal amplification via sequential hybridization. Each fully assembled ampli-

fication structure is contained within 40–50 bp of target RNAwith the capac-

ity for 400-fold signal amplification. Following the QuantiGene ViewRNA

ISHCell Assay, processed filters are mounted to standardmicroscope slides for

imaging [164–166].

3.9. MicroarrayAmicroarray consists of a support onto which hundreds to thousands of dif-

ferent molecular reporter probes are attached or immobilized at fixed loca-

tions in two- or three-dimensional format. Large numbers of targets are

rapidly and efficiently screened using this approach. Microarray probes

can be antibody- or nucleic acid-based [201].

DNA microarrays have been used in numerous applications including

gene expression [202], cell analysis [203], DNA sequencing/fragment

[204], protein [205–207], and genome-wide single-nucleotide polymor-

phism genotyping [208]. Because of this flexibility, microarrays are partic-

ularly useful in the molecular characterization of CTC. Microarray

expression studies, however, require 1–2 mg mRNA from large numbers

of cells (�106–107 cells), thus limiting its application.

3.9.1 Pico profilingPico profiling uses RNA isolation from very small cell populations, cDNA

synthesis and amplification, labeling of cDNA using biotin, and hybridiza-

tion to expression arrays (Affymetrix, Santa Clara, CA, USA). Whole


transcriptome amplification (WTA) is used to generate sufficient cDNA for

microarray expression analysis [209]. WTA is a chemistry based on Tra-

nsplex, a method that generate large amounts of cDNA from nanograms

of RNA based on logarithmic amplification. Transplex performs fragmen-

tation before amplification to overcome differences in amplification effi-

ciencies due to different lengths of transcripts.

This approach requires only 10 cells to generate a secure expression pro-

file. Data are comparable with those produced by standard techniques from

hundreds to millions of cells [209].

3.9.2 Multiplexed PCR-coupled liquid bead arrayPBMC, obtained by gradient centrifugation with Ficoll-Paque PLUS (GE

Healthcare), are subsequently enriched with Immunomagnetic BerEP4-

coated Dynabeads (CELLection Epithelial Enrich, Invitrogen). CTC and

PBMC RNA is then isolated using TRIzol reagent (Invitrogen). Reverse

transcription and PCRmultiplex are performed using primers designed with

SuperScript First Strand Synthesis System for RT-PCR (Invitrogen).

Markou et al. [210] conducted trials using six genes (CK19, ERBB2,

SCGB2A2, MAGEA3, TWIST1, and hydroxymethylbilane synthase). Bio-

tinylated amplicons were hybridized to probe-containing fluorescent micro-

spheres and incubated with streptavidin-phycoerythrin. Captured amplicons

were quantified and beads decoded by Luminex flow cytometry [210].

The results obtained using this approach were comparable to those

obtained using RT-qPCR. Gene expression was not detected in healthy

subjects [185]. CK19, ERBB2, MAGE-A3, SCGB2A2, and TWIST1 were

detected in 27%, 13%, 19%, 11%, and 31% of patients with operable breast

cancer and 65%, 20%, 30%, 20%, and 20% of patients withmetastatic disease,

respectively. In comparison to single trials of RT-qPCR, CK19 has a con-

cordance of 82%, ERBB2 gene of 85%, and TWIST1 of 79%.

Characterization of CTC in real time provides a unique approach to

evaluate tumor phenotype during its disease course [210].

4. MARKERS

Tumor and CTCmarkers are biochemical substances produced by the

cancer as well as those factors produced by the host in response to tumor.

CK has become the best CTC marker for epithelial tumors. Although

the use of multiple markers enhances our ability to detect CTC, none are

ideal due to tumor heterogeneity (Table 6.1).

Table 6.1 CTC markersMarkers used withcytometric techniques Markers used with nucleic acid techniques

CK [187,211–213] ANKRD30A (ankyrin repeat domain 30A) [214,215]

EPCAM [134,216,217] B305D (antigen B305D) [214,215]

ERBB2 [123,218,219] b-HCG (chorionic gonadotrophin) [220–222]

uPAR (plasminogen

activator receptor) [223]

Bmi-1 (B lymphoma Mo-MLV insertion region 1

homolog) [224,225]

CTS [226] c-MET (proto-oncogene met) [185,220,227]

MUC1 [226] CEA [228]

IGF-IR (insulin-like

growth factor 1 receptor)

[229]

CKs [181,185,213,230–233]

EGFR [234–236]

EGP2, epithelial glycoprotein 2 [130]

EPCAM [130,237,238]

GABRP, GABA A receptor pi [214,215]

GalNAc-T (UDP-N-acetyl-D-galactosamine) [220,239]

ERBB2 [184,228,240,241]

MAGEA3 [220]

MUC1 [128,225,242]

MUCL1 (mucin-like 1) [243]

PIP (prolactin-induced protein) [228,244]

PTHrP (parathyroid hormone receptor protein) [245]

SPDEF (SAM pointed domain containing ETS

transcription factor) [244]

TTF1 (trefoil factor 1) [237,238]

TTF3 (trefoil factor 3) [237,238]

SCGB2A1 [228,239,244,246–249]

SERPINB5 [185,250–254]

BIRC5 [254]

miRNA [113,114,255]



5. CLINICAL UTILITY

The presence of occult tumor cells is the major cause of recurrent met-

astatic disease in patients with resection of the primary tumor [256]. Detec-

tion of early recurrence allows for timely initiation of treatment to increase

survival and improve quality of life [160,257–259].

A small percentage (<0.01%) of CTC can successfully start metastatic

colonies. As such, identification of CTC or micrometastatic foci presents

an opportunity for early therapeutic intervention and better risk stratifica-

tion. CTC detection aids diagnosis, prognosis, assessment of recurrence,

and response to anticancer therapy [183].

The prognostic value of CTC may be superior to traditional indices

including site of metastasis (visceral vs. nonvisceral) and estrogen receptor

status. Characterization of CTC provides more information than conven-

tional image methods such as computed tomography and positron emission

tomography to assessing treatment response [135,260].

CTC detection represents a unique minimally invasive approach that

provides real time information on current disease state [261]. Characteriza-

tion of CTC elucidates underlying mechanisms of metastasis and drug resis-

tance, identification of novel diagnostic markers, therapeutic targets, risk

stratification, and development of individualized therapy [262,263].

CTC detection was correlated with decreased progression-free and

overall survival in early [182,184,231,239,264] and advanced breast

cancer [135].

5.1. Early breast cancerThe presence of breast cancer cells or metastatic foci in lymph nodes and bone

marrow is associatedwith poor prognosis in early stage breast cancer [1]. Some

patients with early stage disease developedmetastases following primary tumor

removal. This finding suggested that minimal residual disease could spread and

subsequently develop metastases in distant organs [88]. Stathopoulou et al.

[231] found that CK19 mRNA was an independent prognostic factor for

progression-free and overall survival in both node-negative and node-positive

patients with stage I and II breast cancer. CK19 mRNA-positive cells were

detected in 29.7% of patients with reduced progression-free (p¼0.0007)

and overall survival (p¼0.01).

Xenidis et al. [182] confirmed that detection of CK19 mRNA-positive

cells was predictive of adverse clinical outcome. Adverse prognosis was


associated with ERBB2 negative and triple-negative tumors, but not those

with positive estrogen receptor [184]. CTC count at 4 weeks after chemo-

therapy initiation was a more robust predictor of survival than imaging [265].

Positive CTC ERBB2 mRNA was found in 21% of patients with stage

I and II breast cancer after adjunct chemotherapy [241]. Xenidis et al. [266]

reported that CK19 mRNA-positive CTC were found in 32.7% of patients

at the end of chemotherapy. CTC detection was significantly associated with

increased risk of recurrence and death [241].

The prognostic value of three CTC markers (SCGB2A2, ERBB2, and

CK19) was evaluated in 175 breast cancer patients (stage I–III) before adju-

vant therapy initiation [239]. Cells positive for CK19, MGB1, and ERBB2

mRNA were detected in 41%, 8%, and 29% of patients, respectively. CK19

and SCGB2A2 positivity was associated with shorter disease-free survival

(p<0.001 and p¼0.001) and overall survival (p¼0.044 and 0.034), respec-

tively. ERBB2 mRNA positivity was associated with a shorter disease-free

survival (p<0.001) but not overall survival.

Si et al. [113] suggested that the level of circulating miR-21 andmiR-92a

had predictive value for tumor size and lymph node metastasis in breast

cancer.

Madhavan et al. [114] reported that combinations of miRNA and miR-

200b alone were prognostic for progression-free and overall survival in met-

astatic breast cancer.

5.2. Metastatic breast cancerCTC in peripheral blood in metastatic cancer is an independent marker of

therapeutic efficacy and prognosis. Some studies have evaluated the use of

CTC to identify patients resistant to chemotherapy, so early adjustments

to therapy may be made [260,267]. Three studies independently reported

that high CTC was suggestive of resistance to chemotherapy and that

patients should be changed to a new treatment strategy [268–270].

High numbers of peripheral blood CTC in metastatic breast cancer was

associated with poorer prognosis irrespective of metastasis site, type of ther-

apy and time to recurrence [135,260]. CTC positivity rate was associated

with recently diagnosed metastatic breast cancer (52%) and those receiving

treatment (48%) [135].

Xenidis et al. [266] showed that CK19 mRNA-positive CTC was sig-

nificantly associated with relapse (p<0.001) and disease-related death

(p<0.001). CK19 mRNA-positive CTC was associated with shorter


disease-free survival rates (p<0.001) and a shorter overall survival

(p¼0.001) rate following chemotherapy.

Liu et al. [271] reported that metastatic breast cancer patients with more

than five CTC (baseline, 3–7 weeks and 7–9 weeks after initiation of ther-

apy) were associated with a shorter progression-free survival and a higher

incidence of radiographic progression versus patients with less than

five CTC.

De Giorgi et al. [272,273] compared the prognostic value of CTC at

9–12 weeks after initiating treatment and during systemic therapy, with

FDG-PET/CT in 102 metastatic cancer patients. This study found that

CTC determination could safely predict prognosis.

In a 32-month follow-up, the SUCCESS trial detected more than one

CTC in 9.4% of breast cancer with lymph node metastases prior to chemo-

therapy and 8.7% of patients after chemotherapy [274]. The presence of

more than one CTC before treatment was prognostic of disease-free survival

(p<0.0001) and overall survival (p¼0.023). The persistence of more than

one CTC after chemotherapy was associated with a short duration of

disease-free survival (p¼0.054).

Although several studies have shown the potential usefulness of CTC in

patients with breast cancer, the clinical practice guidelines from the American

Society of Clinical Oncology do not recommend it use for clinical decision-

making. Interestingly, the Southwest Oncology Group Trial S0500 found

DTCweremore easily detected andmore prognostic than CTC [275]. Given

the invasiveness of DTC sampling, CTC detection methodology remains a

promising alternative.

6. CONCLUSION

Analysis ofCTC typically requires a two-step procedure because of their

rarity: enrichment and detection. As such, development of a single step meth-

odology is highly desirable. Unfortunately, there is no standardized approach

forCTCdetectionat this time.Detection ratesvaryconsiderablybasedon tech-

niqueemployedand typeof cancer.Despite tumorheterogeneity, geneexpres-

sion profiles have sufficient sensitivity and specificity to provide a unique and

promising approach for detection and characterization of CTC.

In addition, tumors can transform their cellular characteristics with pro-

gressive loss of epithelial surface markers (EPCAM and CK) and develop-

ment of mesenchymal characteristics (VIM and CDH). These changes

may reflect enhanced aggressiveness. As such, so techniques designed to


detect these changes are critically important in tumor assessment. It is also

likely that gene expression required for metastatic capacity and dissemina-

tion route are different.

Detection of early breast cancer in its early stages is particularly important

for efficacy of treatment and long-term survival. In contrast to traditional

approaches, the use of CTC provides a unique alternative to assess tumor

characteristics. It is clear from the preceding discussion that further research

in this field is warranted. Amore thorough understanding of CTCwill likely

elucidate mechanisms of disease propagation and dissemination, especially in

breast cancer.

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[Advances in Clinical Chemistry] Volume 61 || Markers of Circulating Breast Cancer Cells

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