Lung (2015) 193:157–171
DOI 10.1007/s00408-015-9697-7
REVIEW
Circulating Tumor Cells in Lung Cancer: Detection Methods
and Clinical Applications
Na Yu • Jia Zhou • Fang Cui • Xiaokui Tang
Received: 16 August 2014 / Accepted: 9 February 2015 / Published online: 19 February 2015
Ó Springer Science+Business Media New York 2015
Abstract Circulating tumor cells (CTCs) are tumor cells
that have disseminated from primary and metastatic sites, and
circulate in the bloodstream. Advanced immunological and
molecular-based methods can be used to detect and analyze
the cells with the characteristics of tumor cells, and can be
detected and analyzed in the blood of cancer patients. The
most commonly used methods in lung cancer combine the
processes of immunomagnetic enrichment and immunocy-
tochemical detection, morphology-based enrichment coupled
with reverse transcriptase polymerase chain reaction (RT-
PCR), and RT-PCR alone. CTC analysis is considered a liq-
uid biopsy approach for early diagnosis, risk stratification,
evaluation of curative efficacy, and early detection of lung
cancer relapse. In this review, we discuss the present tech-
niques for analyzing CTCs, and the restrictions of using these
methods in lung cancer. We also review the clinical studies in
lung cancer and discuss the underlying associations between
these studies and their future applications to this disease.
Keywords Circulating tumor cells
Detection methods

Clinical significance
Lung cancer
Review
Introduction
Lung cancer is the most frequently diagnosed cancer and
the leading cause of cancer-related deaths worldwide.
N. Yu
J. Zhou
X. Tang (&)
Department of Respiratory Disease, The First Affiliated Hospital
of Chongqing Medical University, #1 Youyi Rd., Yu-zhong
District, Chongqing 400016, China
e-mail: txk1200@126.com
F. Cui
Department of Clinical Laboratory, The First Affiliated Hospital
of Chongqing Medical University, Chongqing 400016, China
Despite undergoing standardized operative treatments,
many patients with early-stage lung cancer relapse be-
cause of distant organ or tissue metastasis, presumably
due to the undetected spread of the cancer elsewhere in
the body at the time of initial treatment. Currently, there
are no sensitive methods or clinical indexes to recognize
occult micrometastatic tumor cells in patients with lo-
calized lung cancer. In patients with late-stage lung can-
cer, the survival time of this malignant disease is only
minimally prolonged by chemotherapy and/or targeted
drug therapy. Analyzing the genotype and phenotype of
tumor cells may provide essential information that could
prolong survival time and optimize the therapeutic regi-
men of lung cancer patients.
Circulating tumor cells are tumor cells shed from
primary and metastatic sites that circulate in the pe-
ripheral blood, and can be detected by many advanced
immunological and molecular-based methods. The Cell-
SearchTM system is the only diagnostic assay approved
by the US Food and Drug Administration to monitor
patients with metastatic breast, colorectal, and prostate
cancers. In lung cancer patients, CTCs can be recognized
in the peripheral blood depending on the characteristics
of tumor cells and the advanced analysis techniques
used. These cells are identified not only in patients with
distant metastases, but also in patients with localized
early tumors. It is vitally important to develop and op-
timize CTC detection methods, and to summarize the
future management of malignant disease, especially with
regard to risk stratification of adjuvant factors, real-time
monitoring of treatment efficacy, and the development of
new targeted strategies and resistance mechanisms. This
review focuses on the current approaches for CTC de-
tection and analysis, and their clinical applications in
lung cancer.
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158 Lung (2015) 193:157–171

Techniques to Detect CTCs
CTC detection methods comprise the following two parts:
enrichment (isolation) approaches (morphological and im-
munological techniques) and detection (identification) ap-
proaches (cytometric and nucleic acid techniques). The
advantages and disadvantages of the enrichment and de-
tection methods in lung cancer are discussed below and
summarized in Table 1. The optimal method should be
highly sensitive, reproducible, and easy to implement in a
clinical setting.
Enrichment Methods
Enrichment methods are based on the morphological
aspects of CTCs (size or density), or on the selection of
tumor cells using immunoseparation techniques. Most of
these methods include an initial enrichment step such as
density gradient centrifugation, size-based filtration, or
immunomagnetic separation to enhance the probability of
rare cell detection.
Morphology-Based Enrichment Methods
Density-Based Cell Separation
The basis of density-based cell separation is the differential
migration of cells during centrifugation according to their
buoyant density, which results in the separation of different
cell types into distinct layers. Lighter particles including
tumor and mononuclear cells and plasma remain at the top,
and heavier particles including erythrocytes and neu-
trophils migrate to the bottom. Density gradient separation
of CTCs has been performed in lung cancer using Ficoll–
Hypaque or Oncoquick methods to separate CTCs and
mononuclear cells from other blood cells. Compared to the
conventional Ficoll procedure, Oncoquick was developed
to avoid the cross contamination of different layers using a
porous membrane; it was also shown to have higher sen-
sitivity when used for the enrichment of lung cancer cells
[1]. RosetteSepTM (Stem Cell Technologies) uses negative
selection to isolate cells from whole blood, and as such,
that has improved specificity compared to selection using
traditional density gradient sedimentation. In this tech-
nique, bispecific antibodies cross-link white blood cells to
red blood cells to increase their density, thereby allowing
their removal by density gradient centrifugation [2].
Size-Based Cell Separation
Enrichment approaches in which cells are collected by
size-based filtration are also used. Most peripheral blood
leukocytes range from 8 to 11 microns in size, and are in fact
smaller than CTCs derived from epithelial cancers. Thus,
they can easily be removed from the sample by filtration
through a polycarbonate pore membrane. The most com-
monly used membrane microfilter devices have been applied
using several different filtration-based approaches, such as

Table 1 Main advantages and disadvantages of techniques used for CTCs enrichment and detection in lung cancer
Technique Advantages Disadvantages

Enrichment
Immunomagnetic separation
(CellSearch)
Density gradient
centrifugation (Ficoll–
Hypaque or OncoQuick)
Size-based filtration (ISET)
Detection
Cytometric
Nucleic acid based (RT-PCR)
CTC circulating tumor cell, EpCAM epithelial cell adhesion molecule, CTM circulating tumor microemboli, ISET isolation by size of epithelial
tumor cells
Semi-automatic magnetic separation method—easy to
use. Highly reproducibility. High specificity. Fully
validated. FDA-approved method
Simple method. No EpCAM antibody dependent.
Isolates mononuclear cells based on different
density gradient. Non-expensive
Isolates cells based on different size (CTC larger than
WBC). No EpCAM antibody dependent. Effective
method. Tumor cells could be used for the
subsequently analysis. Isolates CTM
Able to assess cell enumeration, morphology and
characteristics. Cells also can be isolated for the
subsequently molecular characterization
Antibody independent. High sensitivity and
specificity, particular in multimarker assay.
Enrichment step is not necessary. Quantitative
analysis
EpCAM antibody dependent. Difficult to extract pure
CTC for further analysis. Low sensitivity.
Expensive
Poor enrichment. Low specificity and sensitivity.
May lost large number of CTCs in processing
Small tumor cells may be omitted. Low sensitivity
and specificity
Detection depends on epithelial or tumor biomarker
expression. No specific CTC marker exists. False-
positive cells may be assessed. Low sensitivity
Difficult to produce. Unable to assess cell
enumeration, morphology and characteristics. The
false-positive is high

123
Lung (2015) 193:157–171
isolation by size of epithelial tumor cells (ISET) [3]. Among
the direct methods used to detect CTCs, ISET technology
allows the substantial enrichment of CTCs in lung cancer
[4]. Moreover, because these cells are available for cy-
topathological analysis, this method may provide specificity
and allow the development of an immunocytochemical ap-
proach for identifying different CTC phenotypes.
Immunomagnetic Separation
One of the most widely applied techniques is immuno-
logical enrichment using specific markers that are ex-
pressed on the surface of CTCs; it involves a magnetic
immunobead and a ferrofluid-based system. There are two
features that are used in current immunological isolation
procedures: epithelial cell-specific markers [epithelial cell
adhesion molecule (EpCAM) and cytokeratins (CK)],
which are generally expressed by tumor cells of epithelial
origin, and tumor-specific markers, which are expressed by
certain types of cancer [5]. These approaches use anti-
bodies that bind target proteins present at the cell surface,
and encompass both positive and negative selection
strategies. Due to the absence of strictly tumor-specific
antigens, epithelial-specific proteins such as EpCAM and
CK have been employed for the positive selection of CTCs,
and antibodies against leukocyte-specific surface antigens
such as CD45 have been used to deplete leukocytes from
the blood sample for negative selection. Immunomagnetic
isolation is performed with monoclonal antibodies coupled
to magnetic microbeads; a strong permanent magnet is
used to isolate CTCs from a leukocyte background by
magnetic force.
Detection
CTC detection methods can be summarily classified into
two groups: cytometric-based and nucleic acid-based ap-
proaches. Cytometric-based techniques use immunocyto-
chemical approaches used to analyze and characterize
different tumor cells; nucleic acid-based techniques detect
DNA or RNA sequences, which have distinct expression
patterns in tumor cells and normal blood components.
Immunocytochemical Approaches
In immunocytochemistry, the cells to be detected are at-
tached to a solid support to allow easy handling. After
fixation, the cells are coated with one or several antibodies.
The choice of appropriate markers is a challenge, because
antigens solely expressed by CTCs are very rare. Anti-
bodies specific to epithelial antigens such as CK and
159
EpCAM are currently the most widely used markers for the
detection of tumor cells of epithelial origin. Antibodies
against epithelial-specific proteins can indicate if and how
many CTCs are present, and leukocyte-specific antibodies
can be used to avoid false positives. Staining can be per-
formed using primary antibodies with detectable tags (such
as a fluorescent molecule), or with tagged secondary anti-
bodies that bind to the primary antibody. These labeling
applications use either enzymatic color reactions or
fluorescence-based techniques for visualization with a light
or fluorescence microscope. Several immunofluorescence-
based techniques are currently used with the aim of for-
mulating a cut-off value of detection. Automated scanning
devices [automated cellular imaging system (ACIS)] sti-
mulate the detection of CTC while decreasing inter-ob-
server discrepancy, and include fiber-optic array scanning
technology, laser-scanning cytometer, and flow cytometry.
Nucleic Acid-Based Techniques
Free circulating DNA can be released from either the pri-
mary or metastatic tumors, or apoptotic CTCs. Thus, the
detection of circulating DNA in the plasma could only
mean the presence of circulating nucleic acids, and not
CTCs, suggesting that circulating DNA in the bloodstream
does not definitely point to the existence of CTCs. An al-
ternative nucleic acid-based strategy is detection of tumor-
associated mRNA by reverse transcriptase polymerase
chain reaction (RT-PCR), which is one of the currently
most widely used methods for detecting CTCs in patients
with lung cancer [6, 7]. Recently, several studies have used
RT-PCR and variations of this technique to detect CTCs in
lung cancer patients by assessing tumor-associated markers
(such as CEA mRNA, CK19 mRNA, LUNX mRNA) ex-
pression in their peripheral blood, and the detection rates
range from 3.63 to 100 % (Table 2). The advantage of
RNA-based approaches for CTC detection is that the
degradation of RNA released from cells in clinical samples
is very rapid; therefore, the origin of detectable RNA
transcripts in a blood sample is considered to be viable
tumor cells. Total RNA is extracted from cells by standard
methods, and transcribed into cDNA using reverse tran-
scriptase. Subsequently, PCR amplification is performed
with the cDNA using primers specific to the transcript of
interest.
To increase sensitivity, an additional PCR reaction with
several primers can be performed on the amplified product
of the first reaction (nested PCR). Both positive and
negative controls are used in these reactions. In addition,
housekeeping genes, such as glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) and b-actin, are used as internal
controls to optimize the quality of RNA and to normalize
the expression levels of target genes.
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 Table 2 CTC enrichment and detection platforms in lung cancer
160

CTC CTC CTC detection CTC Tumor Blood Total CTC counts CTC detection Number of Ref
enrichment enrichment techniques detection stage volume number rate % controls

123
platforms markers markers (ml) of patients (% positivity)

CellSearch Veridex EpCAM CellSpotter analyzer CK 8/18/19, CD45 stages IIIB–IV 7.5 37 C2 per 7.5 ml blood 9/37 (24.32 %) NA [39]
CellSearch Veridex EpCAM Cell track Analyzer II cytokeratin, CD45 stage IV, 10 24 1–24 per 7.5 ml blood 8/24 (33.3 %) NA [32]
recurrence
CellSearch Veridex EpCAM CellTracks Analyzer II CK 8/18/19, CD45 Metastatic NSCLC 20 33 1–19 per 7.5 ml blood 12/33 (36.36 %) NA [40]
CellSearch Veridex EpCAM CellTracks technology cytokeratin, CD45 stage IIIA 7.5 9 C1 per 7.5 ml blood 1/9 (11.11 %) NA [45]
CellSearch Veridex EpCAM Cell track Analyzer II cytokeratin, CD45 SCLC, stages I–IV 7.5 125 C1 per 7.5 ml blood 38/125 (30.4 %) 3/25 (12.0 %) [22]
(florescent (6/9 SCLC, nonmalignant
microscope) 32/116 NSCLC) patients
CellSearch Veridex EpCAM CellSpotter Analyzer cytokeratin 8/18/ stage I 7.5 9 10 per 7.5 ml blood 1/9 (11.11 %) 0/4 healthy [20]
(fluorescent 19, CD45 (before surgery) volunteers
microscope) 1–4 per 7.5 ml blood 3/9 (33.33 %)
( immediately
after surgery)
0/9 (0 %) (10 days
after surgery)
CellSearch Veridex EpCAM CellTracks technology cytokeratin, CD45 advanced NSCLC 10 37 C1 per 7.5 ml blood 28/37 (75.68 %) NA [33]
CellSearch Veridex EpCAM Cell track Analyzer II cytokeratin, CD45 stages I–IV 7.5 30 1–16 per 7.5 ml blood 5/30 (16.67 %) NA [46]
(florescent
microscope)
CellSearch Veridex EpCAM CellTracks technology cytokeratin 8/18/ stage IV 7.5 21 1–21 per 7.5 ml blood 9/21 (42.86 %) NA [34]
19, CD45
CellSearch Veridex EpCAM CellTracks technology cytokeratin, CD45 SCLC 10 51 C2 per 7.5 ml blood 35/51 (68.6 %) NA [23]
(baseline)
49 C2 per 7.5 ml blood 13/49 (26.5 %)
(posttreatment)
37 C2 per 7.5 ml blood 25/37 (67.6 %)
(relapse)
49 C8 per 7.5 ml blood 7/49 (14.3 %)
(posttreatment)
37 C8 per 7.5 ml blood 13/37 (35.1 %)
(relapse)
CellSearch Veridex EpCAM CellTracks technology cytokeratin 8/18/ SCLC 7.5 97 C50 per 7.5 ml blood 41/97 (42.26 %) NA [24]
19, CD45 (baseline)
IMS CD45 ICC Cytokeratin 18, 19 Stages I–IV 7.5 47 C2 per 7.5 ml blood 40/47 (85.11 %) 1/18 (5.56 %) [41]
mRNA healthy donors
and 1/13
(7.69 %)
nonmalignant
pulmonary
tuberculosis
patients
Lung (2015) 193:157–171
 Table 2 continued
CTC CTC CTC detection CTC Tumor Blood Total CTC counts CTC detection Number of Ref
enrichment enrichment techniques detection stage volume number rate % controls
platforms markers markers (ml) of (% positivity)
patients

IMS Cytokeratin 7/8 ICC Pan-cytokeratin Stages I–IV 4 51 NA 26/51 (51.0 %) (6/ 0/20 patients with [47]
18 stage I, 20/33 BLD and 0/20
Lung (2015) 193:157–171

stage II–IV healthy

NSCLC) volunteers
IMS Cytokeratin 7/8 ICC Pan-cytokeratin Stages I–IV 4 35 NA 15/35 (42.9 %) (5/ 0/40, 20 patients [48]
15 stage I, 10/20 with BLD and 20
stage II–IV healthy
NSCLC) volunteers
IMS CD45 Immunofluorescence CK 18 SCLC, stages I–IV 7.5 42 C3 per 7.5 ml blood 20/42 (47.62 %) 0/10 nonmaligant [5]
analysis pulmonary
disease patients
Density gradient Marker independent ICC (fluorescent CK 1/4/5/6/8/10/ stages I–IV 6 –10 71 [2 per 1 9 107 45/71 (63.38 %) NA [49]
centrifugation microscope) 13/18/19, CD45
Density gradient Pan-cytokeratin ICC Lunx and SP-A Stages I–IV 10 32 NA 21/32 65.63 %) 0/32, 16 patients [50]
centrifugation antibody with BLD and 16
and IMS healthy
volunteers
Density gradient CK7/8 antibody ICC CK7/8 Stages I–IV 10 55 1CTC per 5 9 107 29/55 (52.7 %) (7/ 0/50, 25 patients [15]
centrifugation mononuclear cells 21 stage I, 22/34 with BLD and 25
and IMS stage II–IV healthy
NSCLC) volunteers
Density gradient Marker independent RT-PCR Cytokeratin 19 Stages I–IV 10 55 NA 29/55 (52.7 %) (7/ 6/25 (24 %) [15]
centrifugation mRNA 21 stage I patients with
NSCLC, 22/34 BLD and 0/25
stage II–IV healthy
NSCLC) volunteers
LUNX mRNA Stages I–IV 31/55 (56.4 %) (8/ 5/25 (20 %)
21 stage I patients with
NSCLC, 23/34 BLD and 0/25
stage II–IV healthy
NSCLC) volunteers
Density gradient Marker independent Quantitative RT-PCR Survivin mRNA NA 5 68 NA 28/68 (41.18 %) 30 healthy [16]
centrifugation hTERT mRNA 42/68 (61.76 %) volunteers used
to define a cut-off
Cytokeratin-7 28/68 (41.18 %) value for the
mRNA normalization of
TTF-1 mRNA 24/68 (35.29 %) mRNA
combined 56/68 (82.35 %) (at expression
least one marker
positive)
161

123
 Table 2 continued
162

CTC CTC CTC detection CTC Tumor Blood Total CTC counts CTC detection Number of Ref
enrichment enrichment techniques detection stage volume number rate % controls

123
platforms markers markers (ml) of (% positivity)
patients

Density gradient Marker Quantitative RT-PCR EpCAM, MUC1 Stage IA–IIIA 6 61 NA 24/61(39.3 %) 20 healthy [17]
centrifugation independent mRNA (preoperation) volunteers used
16/61(26.2 %) as a calibrator for
(postoperation) normalizing the
mRNA amounts
Density gradient Marker Quantitative RT-PCR Cytokeratin 19 NA 6 55 NA 52/55 (94.5 %) 4/40 (25 %) benign [18]
centrifugation independent mRNA (before the lung disease
initiation of
chemotherapy)
Density gradient Marker Quantitative RT-PCR LUNX mRNA Stages I–IIIA 6 68 NA 40/68 (58.8 %) 42 patients with [35]
centrifugation independent (preoperation) beninge lung
22/68 (32.4 %) diseases used to
(postoperation) set range, defined
as negative
60 20/60 (33.3 %)
(after the
completion of
adjuvant
chemotherapy )
Ficoll–Hypaque Marker ICC Pan-cytokeratin Stages I–III 10 45 The number of cells 11/45 (24.4 %) NA [51]
independent positive for
cytokeratins ranged
from 2 to 10
Ficoll gradient Marker RT-PCR hnRNP-B1 mRNA NA 10 18 NA 14/18 (77.8 %) 0/12 healthy [52]
centrifugation independent Her2/neu mRNA 8/18 (44.4 %) volunteers
Ficoll-Paque Marker RT-PCR Cytokeratin 19 Stages II–IV 5 15 NA 12/15 (80 %) NA [26]
independent mRNA (before
radiotherapy)
8/15 (53 %) (after
radiotherapy)
Ficoll–Hypaque EpCAM antibody Quantitative RT-PCR Cytokeratin 7 Stages IIIB–IV 8 46 NA 14/46 (30.4 %) 6/46 (13.0 %) [8]
and MACS mRNA healthy
volunteers
Cytokeratin 19 4/46 (8.7 %) 0/46 healthy
mRNA volunteers
EGP mRNA 14/46 (30.4 %) 2/46 (4.3 %)
healthy
volunteers
FN1 mRNA 7/46 (15.2 %) 0/46 healthy
volunteers
Lung (2015) 193:157–171
 Table 2 continued
CTC CTC CTC detection CTC Tumor Blood Total CTC counts CTC detection Number of Ref
enrichment enrichment techniques detection stage volume number rate % controls
platforms markers markers (ml) of patients (% positivity)

OncoQuick Marker RT-PCR LUNX mRNA Stages I–IV 10 –20 24 NA 10/24 (41.7 %) (stage NA [1]
independent I–IV NSCLC)
MUC1 mRNA 5/24 (20.8 %) (stage I–
Lung (2015) 193:157–171

IV NSCLC)
Cytokeratin 19 5/24 (20.8 %) (stage I–
mRNA IV NSCLC)
combined 14/24 (58.3 %) (stage
I-IV NSCLC)
StemCell CD45, RT-PCR CLCA2, HMGB3, NA 10 108 (88 NA 49/108 (45.4 %) (at 0/25 healthy [29]
Technologies CD66b, L587S, ASH1 NSCLC least one marker volunteers
CD36 mRNA and 20 positive)
antibodies SCLC)
RosetteSep Human CD45, c - FAST CK 8/18 Stages I–IV 18 –20 17 9–485 per 10 ml blood 16/17 (94.12 %) NA [2]
H2AX
ISET Marker ICC vimentin, pan- Metastatic NSCLC 10 6 5–88 per 5 ml blood 6/6 (100 %) 0/6 healthy [3]
independent cytokeratin volunteers
RT-PCR marker RT-PCR CEA mRNA Stages I–IIIA 5 103 NA 62/103 (60.2 %) 0/15 patients with [25]
independent (preoperation) interstitial
pulmonary
fibrosis who
underwent an
open-lung biopsy
and 0/32 healthy
subjects served as
controls
RT-PCR Marker RT-PCR SCG3 mRNA SCLC, stages I–IV NA 111 NA 51/111 (45.95 %) (36/ NA [53]
independent 67 SCLC, 15/44
stages I–IV NSCLC)
RT-PCR Marker RT-PCR CEA mRNA Stage I NA 29 NA 11/29 (37.9 %) 0/32 healthy [36]
independent (preoperation, by volunteers
video-assisted
lobectomy ) (stage I
NSCLC)
21/29 (72.4 %) (during
and after operation,
by video-assisted
lobectomy)
57 NA 22/57 (38.6 %)
(preoperation, by
open lobectomy)
24/57 (42.1 %) (during
and after operation,
by open lobectomy)
163

123
 Table 2 continued
164

CTC CTC CTC detection CTC Tumor Blood Total CTC counts CTC detection Number of Ref
enrichment enrichment techniques detection stage volume number rate % controls

123
platforms markers markers (ml) of patients (% positivity)

RT-PCR Marker RT-PCR Cytokeratin 19 SCLC, stages I–IV 3 125 (102 NA 57/125 (45.6 %) 5/71 (7 %) healthy [30]
independent mRNA NSCLC, volunteers
PTHrP mRNA 23 SCLC) 81/125 (64.8 %) 5/71 (7 %) healthy
volunteers
LUNX mRNA 35/125 (28 %) 4/71 (5.6 %)
healthy
volunteers
RT-PCR Marker RT-PCR EGFR mRNA Metastatic lung 5 30 NA 17/30 (56.7 %) 4/38 (10.5 %) [54]
independent cancer healthy
volunteers
RT-PCR Marker RT-PCR Cytokeratin 19 Stages I–IV 4 35 NA 20/35 (57.1 %) (7/15 8/20 (40 %) [48]
independent mRNA stage I, 13/20 stage patients with
II–IV NSCLC) BLD and 6/20
(30 %) healthy
volunteers
CEA mRNA 18/35 (51.4 %) (6/15 6/20 (30 %)
stage I, 12/20 stage patients with
II–IV NSCLC) BLD and 5/20
(25 %) healthy
volunteers
RT-PCR Marker RT-PCR CEA mRNA Stage I–IIIA NA 103 NA 62/103 (60.2 %) no healthy [37]
independent (preoperation) volunteers were
29/103 (28.2 %) positive
(postoperation)
Quantitative RT- Marker Quantitative RT-PCR Cytokeratin-7, NA 10 22 NA 22/22 (100 %) (at least 19 healthy [55]
PCR independent EGFR, SCCA, one marker positive) volunteers used
SFTPB mRNA to set range,
defined as
negative
Quantitative RT- Marker Quantitative RT-PCR KRT19 mRNA Stages I–III 2.5 55 NA 21/55 (38.18 %) 12 healthy [56]
PCR independent CEACAM5 mRNA 2/55 (3.63 %) volunteers used
to set range, at
DSG3 mRNA 2/55 (3.63 %) very low or
SFTPA mRNA 2/55 (3.63 %) nondetectable
SFTPC mRNA 2/55 (3.63 %) levels
combined 26/55 (47.27 %) (at
least one marker
positive)
Lung (2015) 193:157–171
 Table 2 continued
CTC CTC CTC detection CTC Tumor Blood Total CTC counts CTC detection Number of Ref
enrichment enrichment techniques detection stage volume number rate % controls
platforms markers markers (ml) of (% positivity)
patients

Nested RT-PCR Marker Nested RT-PCR Cytokeratin 19 Stages I–IV 3 83 NA 30/83 (36.1 %) (5/30 15 healthy volunteers [57]
independent mRNA stage I–II, 25/53 used to set range,
Lung (2015) 193:157–171

stage III–IV NSCLC) defined as negative

Cytokeratin 20 34/83 (41.0 %) (8/30
mRNA stage I–II, 26/53
stage III–IV NSCLC)
CEA mRNA 40/83 (48.2 %) (12/30
stage I–II, 28/53
stage III–IV NSCLC)
combined 61/83 (73.5 %) (18/30
stage I–II, 43/53
stage III–IV NSCLC)
(at least one marker
positive)
Nested RT-PCR Marker Nested RT-PCR TTF-1 mRNA Stages I–III NA 61 NA 22/61 (36.1 %) NA [27]
independent (presurgery)
48 18/48 (37.5 %)
(postsurgery)
Cytokeratin 19 61 26/61 (42.6 %)
mRNA (presurgery)
48 12/48 (25.0 %)
(postsurgery)
Nested RT-PCR Marker Nested RT-PCR SP-D mRNA 26 metastatic and 5 63 NA 33/63 (52.4 %) (24/26 0/30, 15 patients with [58]
independent 37 without with metastases, 9/37 BLD and 15 healthy
metastatic lung without metastases) volunteers
cancer
Nested RT-PCR Marker Nested RT-PCR BJ-TSA-9 mRNA Stage I–IV NSCLC 5 120 NA 68/120 (56.7 %) 1/106 (0.94 %) patients [59]
independent with BLD and 0/80
healthy volunteers
Cytokeratin 19 69/120 (57.5 %) 7/106 (6.6 %) pateints
mRNA with BLD and 3/80
healthy volunteers
Pre-proGRP 42/120 (35.0 %) 6/106 (5.66 %) pateints
mRNA with BLD and 0/80
healthy volunteers
SCC mRNA 29/120 (24.2 %) 9/106 (8.49 %) pateints
with BLD and 3/80
(3.75 %) healthy
volunteers
LUNX mRNA 22/120 (18.3 %) 7/106 (6.6 %) pateints
with BLD and 1/80
(1.25 %) healthy
volunteers
165

123
166 Lung (2015) 193:157–171

[47]

ICC immunocytochemistry, ISET isolation by size of epithelial tumour cells, IMS immunomagnetic separation, MACS magnetic-activated cell sorting system, FAST fiber-optic array scanning
Ref
The sensitivities of these approaches can be evaluated
by analyzing the serial dilutions of a lung cancer cell line in
the peripheral blood of a healthy volunteer. With standard

with BLD and 0/20

with BLD and 0/20

4/20 (20 %) patients

2/20 (10 %) patients

healthy volunteers

healthy volunteers
methods, samples considered positive or negative are based
(% positivity)

on the presence or absence of a PCR product; further

Number of

quantitative analysis cannot be performed. However, the

controls

application of real-time RT-PCR methods now makes the

quantification of cell numbers in clinical samples possible
by comparison with standard curves [8]. Another advan-
stage I, 21/33 stage

stage I, 23/33 stage

28/51 (54.9 %) (7/18

31/51 (60.9 %) (8/18

tage of real-time PCR is that a normal range can be

II–IV NSCLC)

II–IV NSCLC)
CTC detection

established if the assay has been performed for several

controls. Subsequently, a cut-off value for positivity can be
rate %

recognized to improve the specificity of the test.

In addition, fluorescence in situ hybridization (FISH) is
another very common nucleic acid-based technique to ana-
lyze CTCs with prior enrichment for changes in individual
genes, including gene copy number, gene rearrangement, and/
CTC counts

or gene deletion [9, 10]. FISH has previously been considered

as a cogent technique to assess genomic mutations in the
clinic in primary and/or metastatic lesions [11]. This tech-
NA

nique can assess the genomic characteristics of individual

patients
number

CTCs with visual confirmation. After the enrichment step

Total

(ISET or EpCAM-independent immunomagnetic separation),

51
of

ALK gene rearrangement, chromosome 7 and chromosome 8

volume

centromere probes can be detected in CTCs of patients with

Blood

(ml)

NSCLC using FISH analyses, facilitating both diagnostic

4

testing and monitoring of target treatment [9, 10, 12, 13]. Due
to the results of FISH analysis that may not truly represent
cellular phenotype and/or target molecule expression at the
Stages I–IV

protein level, a combination of different detection methods

Tumor
stage

may improve the accuracy of testing results.

Methods for Detecting CTCs in Lung Cancer

Cytokeratin 19

LUNX mRNA
detection

mRNA
markers

Currently, the most widely used methods for detecting

CTC

CTCs in lung cancer combine the processes of immuno-

technology, BLD benign lung diseases, NA not available

magnetic enrichment and immunocytochemical detection

(CellSearchTM System), morphology-based enrichment (-
CTC detection

Nested RT-PCR

density-based cell separation and/or size based on cell

separation) coupled with RT-PCR, and RT-PCR alone
techniques

(Table 2). The CellSearchTM system is the only FDA-ap-

proved device for the enumeration of CTCs in the whole
blood of patients with breast, colorectal, and prostate
independent
enrichment

cancers. Compared to RT-PCR, the major advantages of

the CellSearchTM system are its semi-automation feature,
markers

Marker
CTC

and its high reproducibility, accuracy, linearity, and re-

liability [14]. Although these features are vital to any
Table 2 continued

method to ensure that the results can be validated by dif-

Nested RT-PCR

ferent investigators, such features have been lacking in

enrichment
platforms

previous techniques. Because of the low numbers of CTCs

present in circulating blood, their short half-life, and a
CTC

paucity of functional markers available for their

123
Lung (2015) 193:157–171
identification, the sensitivity of the CellSearchTM system is
very low. In small-cell lung cancer (SCLC) patients, the
detection rates of the CellSearchTM system range from 14.3
to 68.6 %; in non-small-cell lung cancer (NSCLC) pa-
tients, the detection rates range from 0 to 75.7 % (Table 2).
Tissue-specific mRNA may be present although protein-
based assays are negative, and RT-PCR is the most sensi-
tive method for detecting tumor-associated molecular
markers. This sensitivity is remarkably higher than protein-
based assays that involve less sensitive antibody analysis
methods. Paradoxically, RT-PCR is also limited by its
extremely high sensitivity. Sample contamination and il-
legitimate transcription [low-level omnipresent transcrip-
tion of any gene in any cell type or pseudogenes (genes that
lack intrinsic sequences)] may lead to false-positive PCR
amplification results that are indistinguishable from PCR
products derived from intact mRNA. In order to increase
the specificity of RT-PCR in lung cancer, some researchers
have applied density gradient centrifugation [15–18] or
immunomagnetic separation [8] to enrich epithelial cells.
This enriched RT-PCR assay has a higher detection rate of
CTCs than standard RT-PCR, which lacks an enrichment
step (Table 2). The main advantages and disadvantages of
techniques used for CTC enrichment and detection in lung
cancer are listed in Table 1.
Clinical Significance
Large numbers of tumor cells may shed into the circula-
tion, even in the early stages of tumor formation [19]. Yet,
only a minority of these dispersed cells can develop into
overt metastasis. The existence of CTCs in the bloodstream
should not be considered as a definitive indicator of
metastatic disease. To successfully develop metastasis
successfully, CTCs must arrest blood supply, extravasate
into the target organ parenchyma, and proliferate. Tight
vascular wall barriers, adverse living conditions in distant
organs, and the rate-limiting acquisition of organ
colonization functions are some of the impediments to
formation of distant metastasis. These limited metastatic
factors could account for the observation that, despite the
fact that tumor cell shedding occurs during surgical re-
section of primary lung cancer, CTCs disappear from the
circulation after surgery [20]. The detection and enu-
meration of CTCs in lung cancer may serve as an important
biomarker of clinical stage, and prognosis and treatment
predictions [21].
Detection of Lung Cancer and Distant Metastasis
It is likely that the diagnostic value of CTC will play an
important role in detecting lung cancer. For example, there
167
are significantly higher numbers of CTCs in cancer patients
than in benign patients [22], which may be a useful char-
acteristic for the early detection of lung cancer. Among
primary lung cancer patients, SCLC patients showed a
higher CTC enumeration than NSCLC [22–24]. When the
detection rate of CTC was compared with clinical stage,
the higher stage of lung cancer had a higher enumeration of
CTC [22, 25]. Kurusu et al. [25] indicated that the preop-
erative blood samples of patients with clinical stages IIA,
IIB, and IIIA disease (50, 73, and 100 %, respectively) had
a higher incidence of positive carcinoembryonic antigen
(CEA) mRNA than those with stages IA and IB (41 and
36 %, respectively). However, some studies have indicated
that there is no significant difference between CTC status
and clinical stages [26–28]. Wendel et al. [28] evaluated
the association between CTCs and clinical stage in blood
samples from chemotherapy-naı̈ve NSCLC patients using a
fluid phase biopsy approach and found no significant dif-
ference in CTC number among patients with stage IV,
stage III, and stages I/II disease. The presence of CTC in
the blood before therapy does not mean the existence of
advanced or incurable disease; we should detect CTC se-
rially to monitor individual treatment.
Early studies using RT-PCR analyses to detect known
biomarkers (e.g., survivin, hTERT, TTF-1, CK19, PTHrP,
LUNX, CEA mRNA) indicated that patients with
metastatic lung cancer had a significantly higher detection
rate than primary lung cancer patients without distant
metastasis [16, 25, 29, 30]. Katseli et al. evaluated the
association between the mRNA expression of CK19/
PTHrP/LUNX and distant metastases [30]. The authors
found that detection of CK19 mRNA positively correlated
with metastasis to the lymph nodes, brain, bones, and
adrenal glands; detection of PTHrP mRNA closely corre-
lated with the presence of bone metastasis; detection of
LUNX mRNA correlated with lymph node involvement.
However, the sensitivity of RT-PCR analyses for predic-
tion of distant metastasis was not 100 %, suggesting that
imaging examinations [whole-body positron emission to-
mography (PET) scanning, brain magnetic resonance
imaging, computed tomography (CT)] are still necessary,
even when no CTCs are detected. The molecular evidence
of recurrence in lung cancer patients after different treat-
ment regimens has been controversial, due to the fact that
CTCs can shed into the vasculature following treatment
(surgery or radiation). Thus, more studies are needed to
monitor the early recurrence of lung cancer patients.
Detection of Early Relapse
Only a few studies have evaluated the association between
CTC and early recurrence in patients with lung cancer. In a
preliminary study by Sawabata and colleagues [20],
123
168
peripheral blood from nine patients undergoing a lobecto-
my with stage I (IA in 6 cases and IB in 3 cases) NSCLC
was evaluated using the CellSearchTM System. Peripheral
blood samples were collected both during and after the
thoracotomy. One patient was positive for CTC before
surgery, while three were positive immediately after sur-
gery. However, 10 days after surgery, the CTCs disap-
peared in all of these patients. Lung cancer cells may shed
into the circulation during surgery, but due to the afore-
mentioned living conditions, CTCs only persist for a short
time. The small number of events presented in the study
means that the results must be considered preliminary, and
validation is warranted. Nonetheless, the findings suggest
the clinical significance of CTCs in lung cancer. Patients
with undetectable levels of circulating lung cancer cells
had a lower risk of recurrence and mortality; thus, it re-
mains to be determined if these individuals have a better
prognosis, ignoring the influence of other factors, and
should have standard adjuvant therapy withdrawn. Based
on these studies, patients with detectable levels of circu-
lating lung cancer cells may have a higher risk of recur-
rence, and as such, should be offered adjuvant therapy,
while ignoring other prognostic factors.
Prognostic Value of CTCs
Many researchers have evaluated the prognostic value of
CTCs in lung cancer; however, the conclusions of these
studies are controversial. A meta-analysis suggested that
the appearance of CTCs in peripheral blood is associated
with poor prognosis in lung cancer patients [31]. Mean-
while, many studies showed that patients with positive
CTC had a significantly shorter survival time [overall
survival (OS) and/or progression-free survival (PFS)] than
those without [16–18, 23, 24, 27, 32–38]. Naito et al. [23]
indicated that when using 8 CTCs per 7.5 ml blood as the
threshold count, patients with C8 CTCs had a significantly
shorter survival time than the remaining patients with \8
CTCs after therapy in SCLC. Hou et al. [24] analyzed the
association between CTC enumeration and prognosis of
patients with SCLC, and drew the same conclusion using
50 CTCs per 7.5 ml blood as the cut-off value. Krebs et al.
[38] investigated CTC levels before and after chemother-
apy using the CellSearchTM System in NSCLC patients.
When using 5 CTCs per 7.5 ml blood as the threshold
count, patients with \5 CTCs had a mean PFS of
6.8 months, an OS of 8.1 months, and 29 % died in
6 months, whereas patients with C5 CTCs showed PFS of
2.4 months, OS of 4.3 months, and 93 % died in 6 months.
These data indicate that the number of CTCs may be
closely related to the prognosis of patients with NSCLC;
the higher the number of CTCs, the shorter the survival
time of NSCLC patients. However, other studies have
123
Lung (2015) 193:157–171
shown that although the median survival time tends to be
shorter in CTC-positive patients than in CTC-negative
patients, the difference was not significant [39, 40]. When
comparing the results from studies that used samples col-
lected before and after treatments, we found that the sur-
vival outcome was worse for patients with persistently
positive CTC at different time points [35–37]. In addition,
the survival outcome for patients at pre- and post-treatment
time points with persistently negative CTC or for patients
who converted from a CTC-positive to CTC-negative sta-
tus was associated with a longer OS and/or PFS [18]. Due
to the heterogeneity among these studies, further studies
with validated standard techniques should be performed to
evaluate whether treatment-associated variation in CTCs
are related to the prognosis of lung cancer. If such a cor-
relation could be established, novel or established adjuvant
therapies could be supervised, and the dose and duration
could be individualized. Although the same method was
used to detect CTCs in lung cancer in the aforementioned
studies, the thresholds (1, 2, 5, 8, 50/7.5 ml) and results
differed. This illustrates that the existence of CTCs alone
does not have prognostic significance, but rather, other
factors are also important for predicting the outcome of
lung cancer patients, such as the experimental conditions
and epithelial-to-mesenchymal transition (EMT).
Assessment of Therapy
Some studies evaluated the correlation between CTC enu-
meration and radiography (CT scanning or fluorodeoxyglu-
cose-PET), and suggested that CTCs may be related to
disease response rate [33, 41]. Wu et al. [41] conducted a
small-scale study to evaluate the correlation between CTC
number and radiographic appearance (CT scan). CTC
number was determined in 12 patients before and after 2
courses of first-line chemotherapy, followed by CT ex-
amination. Eight of these patients were classified as having
stable disease by CT scanning, and among those patients,
five patients had a reduced CTC count, two patients main-
tained a CTC count of 0, and one patient had a slightly
increased CTC count from 2 to 3 after treatment. For partial
response patients, the CTC count dropped markedly from 52
to 0 in one patient and another two patients had a CTC count
that remained below 2. The only patient whose CTC enu-
meration dramatically increased from 2 to 12 after therapy
was confirmed to have progressive disease by CT scanning.
Punnoose and co-workers [33] also evaluated the association
between CTC count and fluorodeoxyglucose-PET imaging
in patients with NSCLC after the combination of erlotinib
and pertuzumab therapy. The authors found that patients
who showed partial or complete responses had a trend to-
wards higher baseline CTC counts than patients with stable
disease or progressive disease. Meanwhile, the decreased
Lung (2015) 193:157–171
CTC counts upon treatment were significantly associated
with disease response rate; though, one study showed that
CTC count was not associated with disease response rate
[40]. Due to the difference in results, more studies are
needed to determine if there is indeed a correlation between
CTC count and disease response. Using a standardized
imaging method to assess the therapeutic effect may im-
prove the reliability of these results.
Mutation Analysis
Somatic mutations of cancer genes are considered re-
markable markers because they are cancer cell specific, and
such genomic alterations are not affected by changes in
protein marker expression. Recent studies showed that the
mutations in the epidermal growth factor receptor (EGFR)
gene was concordant both in CTCs of blood samples and
matched primary tumor biopsies in patients with NSCLC
[33, 42]; therefore, EGFR mutation analysis is essential to
guide targeted therapy. Maheswaran et al. [42] evaluated
the presence of CTCs and EGFR mutations in patients with
NSCLC. In total, 20 patients had known EGFR mutation
status based on their tumor tissue specimens, the EGFR
mutation status in CTCs was identical to the mutations in
patients’ tumor biopsies in 19 of 20 cases (95 %). T790 M
resistance mutations were detected in two of six patients
(33 %) who had a response to EGFR tyrosinase kinase
inhibitor (TKI) therapy and in 9 of 14 patients (64 %) who
had progression on EGFR tyrosine kinase inhibitor therapy.
Meanwhile, Punnose et al. [33] also analyzed CTCs for
EGFR mutational status by quantitative PCR; despite the
very low sensitivity, the detected mutational status in CTCs
was concordant with tumor biopsies. This indicates that the
detection of CTCs and EGFR mutations may play an im-
portant role in improving the treatment regimens of lung
cancer.
Crizotinib, the ALK inhibitor, is becoming cumulatively
important in targeted therapies for NSCLC patients; thus, it
is necessary to evaluate the presence of ALK gene rear-
rangement in these patients. Pailler et al. [9] evaluated 18
ALK-positive and 14 ALK-negative patients with NSCLC
using a filtration enrichment technique and filter-adapted
fluorescent in situ hybridization. All ALK-positive patients
had four or more ALK-rearranged CTCs per 1 ml of blood,
whereas no or only one ALK-rearranged CTC was detected
in ALK-negative patients. ALK-rearranged CTCs were
detected in five patients during crizotinib treatment. The
levels of CTCs harboring ALK rearrangement and single
ALK native copies decreased in four patients during the
treatment. Evaluating the presence of ALK gene rear-
rangements in CTCs may provide new insights into
monitoring quantitative and qualitative changes in patients
with NSCLC undergoing crizotinib therapy.
169
Conclusion and Future Perspectives
Despite the presence of some ambiguous results, pre-
liminary studies suggest that analyses of CTCs may be
important in the diagnosis and treatment of lung cancer [2,
9, 20, 23, 24, 36, 38, 42]. CTC analyses can be considered
liquid biopsies without invasive tissue biopsies. In addition,
analyses of CTCs may provide broad information for
clinical lung cancer management, including detection, di-
agnosis, and therapy monitoring. It is possible to determine
the changes in CTC number during therapy by repeated
blood sample analyses. However, there are also some
challenges in this field. In current studies, patients show
high variability in sensitivity and specificity which is il-
lustrated by the heterogeneity of study populations and
techniques used. Current cytometric-based and nucleic
acid-based analytical techniques to detect circulating lung
cancer cells are highly sensitive; however, the absence of
lung cancer-associated biomarkers might increase the
false-positive results in patients with lung cancer. During
cancer invasion and metastasis, some cells undergo a
morphological switch from EMT. When using epithelial-
based methods to detect CTCs, false-negative results will
increase in patients with lung cancer [3]. In this respect,
detecting markers of EMT (cytokeratins, E-cadherin, vi-
mentin, neural cadherin) may complement existing ep-
ithelial-based methods, and may improve the positive
detection rate of lung cancer. Due to the abovementioned
reasons, the clinical applications of CTCs have been lim-
ited in lung cancer.
An animal model study suggests that multiple mechan-
isms associated with tumor cell detachment, adhesion, and
suppression of immunogenicity are responsible for the
ability of CTC to enter and survive in the circulation [43];
it is essential for the formation of distant metastases and to
predict recurrent or latent disease; another study indicates
that natural killer (NK) cells play an important role in
preventing metastasis formation because they are able to
decelerate the growth of the primary tumor and kill most of
those CTCs in the blood [44]; it may be useful to improve
and characterize individual treatment of cancer patients.
The abovementioned data about animal model may provide
with clues for the future study of CTC in lung cancer. In
order to improve the clinical value of CTC investigations in
the future, many more studies with standard techniques and
investigations on animal models are needed to establish the
clinical significance of CTC in patients with lung cancer.
Acknowledgments This work was supported by Chongqing Sci-
ence and Technology Commission (cstc2013yykfA10002). The re-
cipient of this Grant is Xiaokui Tang.
Conflicts of interest No potential conflicts of interest were
disclosed.
123
170
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