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Individual Profiling of Circulating Tumor Cell Composition and Therapeutic Outcome in Patients with Hepatocellular Carcinoma

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Circulating tumor cells (CTCs) have been proposed as a monitoring tool in patients with solid tumors. So far, automated approaches are challenged by the cellular heterogeneity of CTC, especially the epithelial-mesenchymal transition. Recently, Yu and colleagues showed that shifts in these cell populations correlated with response and progression, respectively, to chemotherapy in patients with breast cancer. In this study, we assessed which non-hematopoietic cell types were identifiable in the peripheral blood of hepatocellular carcinoma (HCC) patients and whether their distribution during treatment courses is associated with clinical characteristics. Subsequent to few enrichment steps, cell suspensions were spun onto glass slides and further characterized using multi-immunofluorescence staining. All non-hematopoietic cells were counted and individual cell profiles were analyzed per patient and treatment. We detected a remarkable variation of cells with epithelial, mesenchymal, liver-specific, and mixed characteristics and different size ranges. The distribution of these subgroups varied significantly between different patient groups and was associated with therapeutic outcome. Kaplan-Meier log-rank test showed that a change in the ratio of epithelial to mesenchymal cells was associated with longer median time to progression (1 vs 15 months; P = .03; hazard ratio = 0.18; 95% confidence interval = 0.01-2.75). Our data suggest that different CTC populations are identifiable in peripheral blood of HCC patients and, for the first time in HCC, that these individual cell type profiles may have distinct clinical implications. The further characterization and analysis of patients in this ongoing study seems to be warranted.
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Individual Profiling of
Circulating Tumor Cell
Composition and Therapeutic
Outcome in Patients with
Hepatocellular Carcinoma
1
Ivonne Nel*, Hideo A. Baba
, Judith Ertle
,
Frank Weber
§
, Barbara Sitek
, Martin Eisenacher
,
Helmut E. Meyer
, Joerg F. Schlaak
and Andreas-Claudius Hoffmann*
*Department of Medical Oncology, Molecular
Oncology Risk-Profile Evaluation, West German Cancer
Center, University Hospital of Essen, Essen, Germany;
Department of Pathology and Neuropathology, University
Hospital of Essen, Essen, Germany;
Department of
Gastroenterology and Hepatology, University Hospital
of Essen, Essen, Germany;
§
Department of General,
Visceral and Transplantation Surgery, University Hospital
of Essen, Essen, Germany;
Medical Proteome-Center,
Ruhr-University Bochum, Bochum, Germany
Abstract
BACKGROUND AND AIMS: Circulating tumor cells (CTCs) have been proposed as a monitoring tool in patients with
solid tumors. So far, automated approaches are challenged by the cellular heterogeneity of CTC, especially
the epithelial-mesenchymal transit ion. Recently, Yu and colleagues showed tha t shifts in these cell popula -
tions correlated with response and progression, respectively, to chemotherapy in patients with breast cancer. In this
study, we assessed which non-hematopoietic cell types were identifiable in the peripheral blood of hepatocellular
carcinoma (HCC) patients and whether their distribution during treatment courses is associated with clinical charac-
teristics. METHODS: Subsequent to few enrichment steps, cell suspensions were spun onto glass slides and further
characterized using multi-immunofluorescence staining. All non-hematopoietic cells were counted and individual cell
profiles were analyzed per patient and treatment. RESULTS: We detected a remarkable variation of cells with epithelial,
mesenchymal, liver-specific, and mixed characteristics and different size ranges. The distribution of these subgroups
varied significantly between different patient groups and was associated with therapeutic outcome. Kaplan-Meier log-
rank test showed that a change in the ratio of epithelial to mesenchymal cells was associated with longer median time
to progression (1 vs 15 months; P = .03; hazard ratio = 0.18; 95% confidence interval = 0.012.75). CONCLUSIONS:
Our data suggest that different CTC populations are identifiable in peripheral blood of HCC patients and, for the first
time in HCC, that these individual cell type profiles may have distinct clinical implications. The further characterization
and analysis of patients in this ongoing study seems to be warranted.
Translational Oncology (2013) 6, 420428
Address all correspondence to: Andreas-Claudius Hoffmann, MD, Assistant Professor of Experimental Oncology, Department of Medical Oncology, Molecular Oncology Risk-Profile
Evaluation, West German Cancer Center, University Hospital Essen, Hufelandstrasse 55, 45147 Essen, Germany. E-mail: hoffmann@more-oncology.com
1
This project is financed by funds of the Ziel 2Programm NRW 2007-2013, Bio.NRW, the European fund for regional development (EFRE, Europäischer Fonds für regionale
Entwicklung,”“Investition in unsere Zukunft), and funds from the Ministry of Innovation, Science and Research of North Rhine-Westphalia, Germany; this project was also
presented at the Advances in Circulating Tumour Cells (ACTC) Conference in Athens in 2012. No author has any conflict of interest that is relevant to the manuscript.
Received 10 March 2013; Revised 30 April 2013; Accepted 2 May 2013
Copyright © 2013 Neoplasia Press, Inc. All rights reserved 1944-7124/13/$25.00
DOI 10.1593/tlo.13271
www.transonc.com
Translational Oncology
Volume 6 Number 4 August 2013 pp. 420428 420
Introduction
Hepatocellular carcinoma (HCC) is associated with a poor prognosis
and is among the five most common malignancies worldwide with an
increasing incidence [1,2]. Curative therapeutic options are limited
to early stages and include mostly resection or orthotopic liver trans-
plantation if patients present with cirrhosis [3,4]. High recurrence
rates after resection and liver transplantation, most likely because
of minimal residual disease [5 ,6], and the fact that the majority of
patients are diagnosed in an advanced stage make palliative, often
localized approaches including selective internal radiation t herapy
and transcatheter arterial chemoembolization necessary [7,8]. Up to
now, there are no reliable early markers of relapse or response to
surgical or interventional therapy. Serum-based markers like alpha-
fetoprotein (AFP), des-gamma-carboxyprothrombin, or the lectin 3
fraction of AFP (AFP-L3) are incapable of predicting the clinical out-
come with high accuracy and reproducibility [9]. Tissue-derived
molecular markers lack the possibility of monitoring the patient during
or after treatment, because this would require repeated biopsies and
hence increased risks for the patient. Therefore, the development of
minimally invasive diagnostic methods is necessary.
Circulating tumor cell (CTC), detected in the peripher al blood
of HCC patients, may represent a possible solution for this diag-
nostic dilemma. Though these cells have been frequently described
in breast and lung cancers [10 12], only few studies reported on
CTC in HCC patients using indirect methods like quantitative real-
time reverse transcriptionpolymerase chain reaction or direct visuali-
zation of circulating epithelial cells [1317]. The main obstacle to
the broad clinical application of available automated CTC detection
methods i s the high plasticity and variability of these cells among
others due to the epithelial-mesenc hymal transition (EMT ) as has
been very recently shown by Yu and colleagues in breast cancer [18].
In this ongoing study, we wanted to scrutinize the hypothesis that
a large variety of circulating non-hematopoietic cells exist in the pe-
ripheral blood of patients with HCC and that these cell types change
during treatment and whether these changes may implicate resistance
to therapy.
Materials and Methods
Study Population and Informed Consent
Patients with solid malignancies that received anticancer treatment
in our hospital were consecutively included in this study after agree-
ing and signing a written informed consent in accordance with the
requirements of our institutions board of ethics (Internal Reference
No. 12-5047-BO). For this feasibility study, we tested the first 11 patients
with a follow-up of at least 6 months. There were no inclusion criteria
besides evaluable progression time and either being resected or receiv-
ing local ablative or systemic treatment. Patients with watch and wait
were assessed but not included in outc ome-related statistics at the
time being. The clinicopathologic data of the patients used for CTC
quantification is listed in Table 1.
Blood Samples
Twenty milliliters of citrated peripheral blood from HCC patients
was drawn during treatment visits in the outpatient unit of our liver
tumor center, stored at room temperature, and processed within
24 hours after collection. We employed a negative selection strategy
to enrich and detect CTC. Hematopoietic cells were depleted using
anti-CD45 immunomagnetic beads leading to a bead-free cell sus-
pension that was then used for CTC type detection by immuno-
fluorescence staining against various epithelial [e.g., pan-cytokeratin
(pan-CK)] and mesenchymal markers (e.g., vimentin, N-cadherin).
Cell Culture and Spiking Experiments
HCT 116 and HepG2 cell lines were obtained from American
Culture Type Collection (ATCC, Rockville, MD) and maintained
according to ATCC guidelines. For method validation, we used
20 ml of peripheral blood from healthy donors spiked with 100
HCT 116 cells. Spiked blood samples were processed using density
gradient centrifugation and immunomagnetic enrichment strategy as
described below. HepG2 ce lls were mixed with peripheral blood
mononuclear cells (PBMNCs) from healthy donors and served as posi-
tive and negative controls for immunofluorescence staining. Gastro-
intestinal stromal tumor cells GIST 882 were a kind gift from Dr Bauer
(Department of Medical Oncology, University Hospital Essen).
Aliquots of GIST 882 cells were washed, resuspended in phosphate-
buffered saline (PBS), and spotted onto glass slides for subsequent
immunofluorescence staining against mesenchymal markers.
Sample Preparation
Isolation of P BMNCs using density gradient centrifugation.
Twenty milliliters of citrated peripheral blood was diluted with 10 ml
of PBS and poured into a Leucosep tube (Greiner Bio-One, Fricken-
hausen, Germany) prepared with 16-ml Ficoll-Paque (GE Healthcare,
Buckinghamshire, Great Britain) below the porous barrier. After density
gradient centrifugation at 1600g at 20°C for 20 minutes without brake,
the PBMNCs containing interphase above the barrier were transferred
into a new tube, washed with 50 ml of PBS containing 0.5% BSA,
and centrifuged at 3 00g at 20°C for 10 minute s. Subseque ntly, the
supernatant was removed completely.
Table 1. Patient Demographics for Quantification.
Demographic Patients (n = 11)
No. %
Tumor size (mm)
Median 49
Range 30170
Child status
A982
B218
Cirrhosis 7 64
Chronic hepatic viral infection
HBV 2 18
HCV 4 36
Age
Median, years 70
Range 4285
Therapy
Watch and wait 3 27
Selective internal radiation therapy 5 45
Transcatheter arterial chemoembolization 1 9
Resection 1 9
Nexavar 1 9
Response
Stable disease 4 36
Progressive disease 4 36
Time to progression (months)
Median 3
Range 019
Translational Oncology Vol. 6, No. 4, 2013 Circulating Tumor Cell Profiles in Liver Cancer Nel et al. 421
Tumor cell enrichment using immunomagneti c beads. Washed
PBMNCs were resuspended in 1 ml of PBS containing 0.1% BSA
and incuba ted with 25 μlofanti-CD45coated immunomagnetic
Dynabeads (Invitrogen, Carlsbad, CA) for 30 minutes. Hematopoietic
cells except erythrocytes, platelets, and their precursor cells were bound
to beads and separated by a magnetic particle processor (King Fisher
mL; Thermo Fisher, Waltham, MA). The remaining cell suspension
included bead-free pre-enriched tumor cells and was spun onto two
glass slides per sample using the C ell Spin II centrifuge (Tharmac,
Waldsolms, Germany), air dried, and subsequently fixated with 96%
etha nol. Slides were stored at 4° C until subjected to immunocyto-
chemical staining. For assessment of the depletion rate, PBMNCs were
stained with trypan blue and enumerated using an automated cell
counter (Countess; Invitrogen) before and after immunomagnetic
separation. The results indicated an avera ge CD45 depletion of
73%. By increasing the concentration of immunomagnetic beads,
the depletion rate could be increased up to 95%, though with an
increasing damage in respect to cell morphology.
Identification of Spiked HCT 116 Cells after Density Gradient
Centrifugation and Immunomagnetic Enrichment Using
Immunocytochemical Labeling
Detection of HCT 116 cells was performed using the Epimet kit
(Micromet, Munich, Germany). The identification of epithelial cells
is based on the reactivity of the murine monoclonal antibody (mAb)
A45-B/B3, directed against a common epitope of CK polypeptides.
The kit uses Fab fragments of the pan-mAb conjugated with alkaline
phosphatase molecules. The method includes permeabilization of the
cells by a detergent (5 minutes), fixation by a formaldehyde-based
solution (10 m inutes), binding of the conjugate mAb A45-B/B3
alkaline phosphatase to cytoskeletal CK (45 minutes), and formation
of an insoluble red reaction product at the binding site of the specific
conjugate (15 minutes). Subsequently, the cells were counterstained
with Mayers hematoxylin for 1 minute and finally mounted with
Kaisers glycerin/gelatin (Merck, Darmstadt, Germany) in Tris-EDTA
buffer (Sigma, Deisenhofen, Germany). A conjugate of Fab fragment
served as a negative control. For each test, a positive control slide with
the colon carcinoma cell line HCT 116 was treated under the same
conditions. The microscopic evaluation was carried out using the
ARIOL System (Applied Imaging, Newcastle upon Tyne, United
Kingdom) in the Department of Gynecology and Obstetrics (Essen,
Germany) according to the International Society of Hematotherapy
and Graft Engineering (ISHAGE) evaluation criteria and the dissemi-
nated tumor cells (DTC) consensus [19,20]. This automated scanning
microscope and image analysis system consists of a slide loader, camera,
computer, and software for the detection and classification of cells of
interest based on particular color, intensity, size, pattern, and shape.
Identification of CTC Subtypes Using
Multifluorescence Labeling
Immunofluorescence staining of epithelial, mesenchymal, liver-
derived, and hematopoietic cells was carried out in the CD45-depleted
pre-enriched tumor cell suspension. Briefly, the staining method included
fixation of the cells in 4.5% paraformaldehyde for 15 minutes, washing
in PBS, permeabilization with Perm/Wash Buffer (BD Biosciences,
Franklin Lakes, NJ) for 10 minutes, washing in PBS, blocking of un-
specific antibody reactions by incubation with blocking solution contain-
ing 5% BSA for 30 minutes, binding of primary antibodies (final
concentration, 5 μg/ml) either ASGPR1 rabbit mAb (ab42488; Abcam,
Cambridge, United Kingdom) or pan-CK guinea pig polyclonal anti-
body (ABIN126062, antibodies-online) and epithelial cell adhesion
molecule (EpCAM, E144) rabbit mAb (ab32392; Abcam) or vimentin
(EPR3776) rabbit mAb (2707-1; Epitomics, Burlingame, CA) or
N-cadherin (EPR1792Y) rabbit mAb (2019-1; Epitomics) for CTCs
and anti-CD45 (MEM-28) mouse mAb (ab8216; Abcam) for hemato-
logic cells overnight at 4°C, washing in 0.1% Tween, binding of second-
ary antibodies (fluorescein isothiocyanateconjugated AffiniPure goat
anti-rabbit and Cy3-conjugated AffiniPure goat anti-mouse or Alexa
Fluor 647 conjugated AffiniPure F(ab)2 fragment goat anti-guinea
pig; Jackson ImmunoResearch, Hamburg, Germany) for 30 minutes at
37°C, and washing in 0.1% Tween. Subsequently, cells were stained with
46-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich,
St Louis, MO) for 10 minutes, mounted with anti-fading medium
(Invitrogen), and stored in the dark until evaluation. For each test, a posi-
tive control slide with a mixture of human PBMNCs and the HCC cell
line HepG2 was treated under the same conditions. Gastrointestinal
stromal cells (GIST 882) were used as positive control for mesenchy-
mal markers. Microscopic evaluation was carried out using the digital
Keyence BZ9000 (Biorevo, Osaka, Japan) all-in-one fluorescence
microscope with integrated camera and BZ-Analyzer Software. We used
Figure 1. (A) Cultured HCT 116 cells were harvested, washed, and spun onto glass slides using the Cell Spin II centrifuge. Subsequently,
they served as positive control for immunocytochemical staining against pan-CK (pink) using alkaline phosphatase. (B) Manual microscopic
detection of spiked HCT 116 cells in peripheral blood from healthy donors enriched with density gradient centrifugation and negative
enrichment through anti-CD45 immunomagnetic beads and stained against pan-CK. Pan-CKpositive cells are pink. Little brown round
particles are immunomagnetic beads. (C) Detection of spiked HCT 116 cells using the ARIOL-SL automated scanning system resulted
in a recovery rate of 100% after density gradient centrifugation and 43% after depletion of hematopoietic cells using anti-CD45coated
immunomagnetic beads. (D) Detection of pan-CKpositive cell in peripheral blood from HCC patient using the ARIOL-SL System.
422 Circulating Tumor Cell Profiles in Liver Cancer Nel et al. Translational Oncology Vol. 6, No. 4, 2013
pseudocolors to depict cells. Stained slides were manually examined and
CTCs were detected within the same areas, each consisting of 10 visual
fields usin g a ×20 magnif ication on both slides. Samples from five
healthy donors were processed and examined under the same conditions
to define a cutoff value for false-positive events.
Statistical Analysis
Statistical tests were performed according to previously published
studies by our group [2123]. Spearman rank correlation was used
to examine associations between CTC subtypes and clinical charac-
teristics of the patients. Associations of the cell type ratios and time
to progression (TTP) were tested by the Kaplan-Meier method. Sur-
vival differences between patients with high and low cell type ratios
were analyzed by the log-rank test. The level of significance was set to
P <.05.AllP values were based on two-sided tests. All statistical analy-
ses were performed using the Software Packages SPSS for Windows
(Version 19.0; SPSS Inc, Chicago, IL) and Medcalc, Version 12.3.0
(Mariakerke, Belgium).
Results
Enrichment Procedure
Analysis of the spiking experiments with 20 ml of whole blood
and 100 epithelial HCT 116 cells using the automated scanning
Figure 2. (A) Positive control consisting of PBMNCs mixed with HepG2 cells; stained against DAPI (blue) and epithelial or liver-specific
markers: pan-CK (red) and CD45 (green); EpCAM (yellow) and CD45 (green); ASGPR1 (red) and CD45 (green). PBMNCs were positive for
CD45 (green) and HepG2 cells were positive for pan-CK (red); EpCAM (yellow) and ASGPR1 (red). (B) CTC isolated from HCC patients
stained against DAPI (blue) and pan-CK (red)/CD45 (green) an d EpCAM (yellow)/CD45 (green) and ASGPR1 (red)/ CD45 (green). Cells
marked with a white arrow were considered as CTCs. They showed a DAPI-positive (blue)/CD45-negative staining and were positive
for pan-CK (red) or EpCAM (yellow) or ASGPR1 (red).
Translational Oncology Vol. 6, No. 4, 2013 Circulating Tumor Cell Profiles in Liver Cancer Nel et al. 423
microscope ARIOL-SL revealed a recovery rate of 100% after density
gradient centrifugation. Subsequent depletion with immunomagnetic
beads led to a significant lower recovery rate of 45% (Figure 1, AC).
Furthermore, we were able to detect CK-positive cells in the peripheral
blood of HCC patients using this detection system (Figure 1D).
Immunofluorescence-based Identification of CTC Subtypes
For the investigation of cellular subtypes, a multistaining method
was required to detect various epithelial, mesenchymal, and hemato-
poietic markers and to characterize different cell types. Therefore, we
used multifluorescence staining for CTC subtype detection in HCC
patients. A mixture of PBMNCs from a healthy donor spiked with
epithelial cells from the HCC cell line HepG2 was used as positive
control and n egative c ontrol. Hematopoietic cells showed positive
staining against CD45, whereas HepG2 cells were CD45-negative.
HepG2 cells stained positive against the epithelial markers pan-CK
and EpCAM and against the liver-specific ASGPR1 (Figure 2A).
When examining samples from HCC patients, objects that showed
a positive nuclear staining with DAPI, a negative staining for CD45,
and a positive staining against pan-CK, EpCAM, or ASGPR1 were
captured and considered as tumor cells (Figure 2B). We used GIST
882 cells as positive control for staining against the mesenchymal
markers N-cadherin and vimentin (Figure 3A). In HCC blood sam-
ples, we detected cells with mesenchymal features such as N-cadherin+/
CD45 and vimentin+/CD45 after negative isolation. We also de-
tected cells showing both epithelial and mesenchymal characteristics
such as N-cadherin+/CK+/CD45 and vimentin+/CK+/CD45 (Fig-
ure 3, B and C). In addition, we found cells that stained positive for
pote ntial markers of CTC an d CD45 such as pan-CK+/EpCAM+/
CD45+ as well as pan-CK+/EpCAM/CD45+ cells (Figure 4A [24]).
CTC profiles of each patient were examined and a ratio of mesenchymal
to epithelial cells was conceived (Figure 4B). Although we detected a
variety of cellular subtypes, we chose to summarize the total amount of
N-cadherinpositive and vimentin-positive/CD45-negative cells in
proportion to the total amount of CK-positive cells, respectively, after
negative enrichment using CD45 depletion. We normalized the enu-
merated potential CTC against the total PBMNC number detected in
the DAPI cha nnel in each visual field and expressed th e number of
CTC per 1000 PBMNCs (Figure 4C). Analysis of samples from healthy
donors revealed a cutoff for false-positive events at six CK-positive, one
N-cadherinpositive, and two vimentin-positive/CD45-negative cells
per 1000 PBMNCs after CD45 depletion.
Subtypes and Clinical Outcome
Spearman rank test resulted in a by trend significant association of
TTP with the N-cadherin+/CK+ ratio (P = .06) and the vimentin+/
CK+ ratio (P = .07), whereas there was no significant correlation of
TTP of the total amount of CK-positive, N-cadherinpositive, and
vimentin-positive cells. The total amount of N-cadherinpositive and
vimentin-positive cells was associated to the amount of CK-positive
cells (P =.05andP = .1). Interestingly, 7 of 11 patients had cirrhosis,
but there was no association to TTP (P = .18). Though, the total
amount of CK-positive cells showed a by trend significant correlation
to cirrhosis (P =.1;Figure5A) and the ratio of N-cadherin+/CK+ cells
Figure 3. (A) Immunofluorescence staining of GIST 882 cells as positive control for N-cadherin (yellow) and vimentin (yellow), respectively.
(B) Overlay picture of a CTC from an HCC patient showing both epithelial and mesenchymal features. This cell stained positive for pan-CK
(red), N-cadherin (green), and DAPI (blue). (C) CTC subtypes showing mesenchymal and/or epithelial features.
424 Circulating Tumor Cell Profiles in Liver Cancer Nel et al. Translational Oncology Vol. 6, No. 4, 2013
was significantly correlated to cirrhosis (P =.03;Figure5B). The amount
of epithelial CK+ cells (P = .1) and t he ratio of N-cadherin +/CK+
cells (P = .1) show ed a by trend significant correlation to the Child
status (Figure 5C).
To test whether the N-cadherin+/CK+ ratio or the vimentin+/CK+
ratio potentially divided patients according to their chance of a pro-
longed TTP, we used the Kaplan-Meier log-rank test, resulting in a
significant association of both ratios with TTP. A vimentin+/CK+
ratio higher than 0.5, meaning that there were half as many vimentin-
positive cells as CK+ cells, was associated with a longer median TTP
[1 vs 15 months; P = .03; hazard ratio (HR) = 0.18; 95% confidence
interval = 0.012.75]. A N-cadherin+/CK+ ratio lower than 0.1 re-
sembled an association to shortened TTP (1 vs 15 months; P =.03;
HR = 0.19; 95% confidence interval = 0.012.75; Figure 5D).
Discussion
In this study, we used multi-immunofluorescence staining to detect
a significant variety in interindividual as well as intraindividual CTC
characteristics. We measured epithelial cells, staining positive for
pan- CK (+DAPI) but negative for EpCAM, and CD45 as well as
cells staining positive for both CK and EpCAM while negative for
CD45. We also detected CTC with mesenchymal properties such as
vimentin and N-cadherin as well as cells with both epithelial and
mesenchymal features, remarkably with changing ratios even during
local ablative treatment of patients. It has very recently been described
by Yu and colleagues that CTC undergo EMT during treatment and
that these changes, and not only the absolute numbers of certain sub-
groups, correlate well with response and resistance to cytotoxic treat-
ment, respectively [25]. The detection of CTC showing mesenchymal
phenotype was also reported by Gradilone and colleagues who char-
acterized CTC for CK and markers of EMT and found the gain of
mesenchymal markers in CTC to be correlated to patient prognosis
in a follow-up of 24 months [26,27]. Their data showed that the pres-
ence of mesenchymal markers on CTC more accurately predicted a
poor prognosis than the expression of CK alone. Here, we were able
to confirm that the presence of mesenchymal cells correlates to survival
in HCC patients. However, our results in this study group indicated
that an increase in epithelial cells was associated with worse treatment
outcome in patients with HCC, which leads us to hypothesize that
epithelial cells may be the driver of aggressiveness in this study popu-
lation. Supporting these findings, we also revealed that the total
amount of CK+ cells was higher in patients with cirrhosis and in
patients with a worse Child status. These contradicting results may
be explainable by the different entities (breast vs hepatocellular cancer),
different treatment approaches, or a limited sample size. The shift
from mesenchymal to epithelial cell profiles was significantly correlated
with shortened TTP in both N-cadherin and vimentin to CK ratios,
respectively, and indicated that our data might be valid despite the
low patient number.
Figure 4. (A) Cells considered to be CTCs are indicated with a white arrow. This figure depicts the heterogeneity of detected CTC in one
single HCC blood sample. Amo ng hematopoietic CD45-pos itve cells, it shows two CTCs that were b oth pa n-CKpositive (red) and
CD45-positive (green), while one of them was also EpCAM-positive (yellow). All cells showed a positive nuclear staining (DAPI, blue).
(B) Example of individual morphologic profile showing one epithelial (CK-positive) and three mesenchymal (N-cadherinpositive) cells.
(C) CTC subtypes were summarized, normalized against the total amount of cells in the DAPI channel per visual field, and expressed in
potential CTC per 1000 PBMNCs after enrichment.
Translational Oncology Vol. 6, No. 4, 2013 Circulating Tumor Cell Profiles in Liver Cancer Nel et al. 425
Remarkably, in our study, epithelial and mesenchymal cells were
detectable in the peripheral blood of almost all patientsin different
proportionswhereas only some of these patients (4/11; 36%) pre-
sented cells with both features on the same cell potentially indicating
different stages of EMT. However, this patient group is too small
to answer this question conclusively at this point. Additionally, a
distinct proportion of cells stained positive for pan-CK and CD45
as well as for EpCAM, pan-CK, and CD45, a phenomenon already
described by Yu and colleagues [24]. The additional CD45+ staining
may not be exclusive for hematopoietic cells but may hypothetically
be acquired during dormant s tay in the bone marrow or through
effects comparable to trogocytosis, i.e., transfer of membrane pro-
teins [28]. Ther efore , depletion of CD45-positive cells might lead
to a loss of cells of interest during the negative enrichment proce-
dure. Moreover, the retrieval was significantly lower after immuno-
magnetic depletion compared to density gradient centrifugation alone
as shown by our spiking experiments. Though when anticipating
CTC isolation from blood recapturing spiked cultured cells it remains
unclear to which extent these experiments reflect the in vivo situation.
CD45 depletion will be omitted in the following studies to obtain an
increased CTC recovery rate. After determining the total amount of
cells, samples will be spun onto the required amount of slides with
maximum of 5 × 10
6
cells per slide that will lead to a higher number
of sl ides with increased staining o ptions allowing to investigate a
wider range of cell type characteristics. Even though the depletion
rate was not very effective, we chose to proceed with the protocol to
diminish hematopoietic cells and to be able to overlook different cell
types within blood samples. Moreover, our method is based on cell
type ratios rather than a bsolute cell numbers a ssuming that a loss
of potential CTC might not affe ct the proportion of their CD45-
negative subtypes. These question s need to be addressed in sub-
sequent investigations analyzing a higher number of blood samples
and multiple CTC subtypes also inc luding endothelial, stem cell,
and other characteristics.
Figure 5. (A) The total amount of CK+ cells/1000 PBMNCs was higher in patients with cirrhosis compared to patients without cirrhosis
(P = .1). (B) The ratio of N-cadherin+/CK+ cells was also correlated to cirrhosis. Patients with cirrhosis had a higher total amount of
CK+ cells that led to a lower ratio of N-cadherin+/CK+ cells compared to patients without cirrhosis (P = .03). (C) The ratio of N-cadherin+/
CK+ cells was correlated to the Child status (P = .1). (D) Association of high vs low N-cadherin+/CK+ CTC ratio with TTP employing Kaplan-
Meier log-rank test (P = .03; HR = 0.18).
426 Circulating Tumor Cell Profiles in Liver Cancer Nel et al. Translational Oncology Vol. 6, No. 4, 2013
In contrast to CTC in other entities being commonly described
to have a large cellular size and a high nuclear to cytoplasmic ratio
[14], our results confirm a previous report from a patient with breast
cancer by Marrinucci and colleagues showing a wide range of signal
intensities and a variety of cell sizes [29,30]. This is of relevance
because the isolation by size of epithelial tumor cells using the ISET
System (Rarecells, Paris, France) is one of the few major (semi)
automated approaches used for detecting CTC. Vona et al. used
ISET in patients with HCC to evaluate its clinical impact. Fifty-
two percent of a total of 44 patients had detectable CTC and their
prevalence was significantly correlated with the presence of multi-
focal tumors, portal tumor thrombo sis, and Child-Pu gh cla ss B/C
[15]. In a study of Schulz e and colleague s, the CellSearch System
was applied to detect EpCAM-positive CTC in the blood of HCC
patients resulting in a detection rate of 45% (14 of 31 HCC patients)
and a significant correlation between the detection of epithelial
CTC and pathologic AFP values [17]. Xu et al. reported the devel-
opment and validation of an EpCAM-independent magnetic cell
separation system mediated by the interaction of the ASGPR1 exclu-
sively expressed on hepatocytes with its ligand. CTCs were then
identified by Hep-Par-1 staining. An average of 24 ± 19 CTCs per
5 ml of blood was detected in 81% of HCC patients, suggesting that
the variation ranged from one to nine tumor cells per millilite r in
the examined patients. Therefor e, with this study, we wanted to
show that the individual cell composition and especially changes in
this personal cell profile implicate alternating therapeutic responses,
pote ntially with more sensitivity than currently available. F urther-
more, these changes may lead to specific treatments, aiming at distinct
CTC profiles.
Taken together, o ur data support the hypothesis that different
CTC populations are identifiable in the peripheral blood of patients
with HCC and that these individual cell type profiles may have dis-
tinct clinical implications. This method offers an opportunity to detect
changes in the composition of circulating non-hematopoietic cells in
the blood of patients with HCC and, therefore, to create an individual
profile of each patient, which might help to predict patient outcome
and potentially to select the appropriate treatment. Further studie s
including systemic treatments of HCC seem to be warranted.
Acknowledgments
We thank Sabine Kasimir-Bauer and her team (Department of Gyne-
cology and Obstetrics, University Hospital Essen) for supporting the
validation of our method using the ARIOL-SL System. We also thank
Karl Lang (Institute of Immunology, University Hospital Essen) for
constantly granting us access to his Keyence all-in-one fluorescence
microscope. Furthermore, we thank Sebastian Bauer and his team
(Department of Medical Oncology, University Hospital Essen) for
the supply of GIST 882 cells.
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Purpose To investigate the role of circulating rare cells (CRCs), namely, circulating tumor cells (CTCs) and circulating endothelial cells (CECs), in aiding early intervention, treatment decision, and prognostication in bladder cancer. Methods A total of 196 patients with pathologically confirmed bladder cancer, namely, 141 non-muscle invasive bladder cancer (NMIBC) and 55 muscle invasive bladder cancer (MIBC) patients. There were 32 patients who received cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC). Subtraction enrichment combined with immunostaining-fluorescence in situ hybridization (SE-iFISH) strategy was used for CTC/CEC detection. Kaplan–Meier analysis and Cox regression were used to evaluate the overall survival (OS) and recurrence-free survival (RFS). Receiver operator characteristic analysis was used to discriminate NAC sensitivity. Results CTCs and CECs were related to clinicopathological characteristics. Triploid CTCs, tetraploid CTCs, and total CECs were found to be higher in incipient patients than in relapse patients ( P = 0.036, P = 0.019, and P = 0.025, respectively). The number of total CECs and large cell CECs was also associated with advanced tumor stage ( P = 0.028 and P = 0.033) and grade ( P = 0.028 and P = 0.041). Remarkably, tumor-biomarker-positive CTCs were associated with worse OS and RFS ( P = 0.026 and P = 0.038) in NMIBC patients underwent TURBT. CECs cluster was an independent predictor of recurrence in non-high-risk NMIBC patients underwent TURBT (HR = 9.21, P = 0.040). For NAC analysis, pre-NAC tetraploid CTCs and small cell CTCs demonstrated the capability in discriminating NAC-sensitive from insensitive patients. Additionally, tetraploid CTCs and single CTCs elevated post-NAC would indicate chemoresistance. Conclusion CTCs and CECs may putatively guide in diagnosis, prognosis prediction, and therapeutic decision-making for bladder cancer.
... It has been widely correlated with disease aggressiveness, resistance to therapy and decreased PFS and OS, and it may underlie the biology of tumor dissemination and treatment resistance [56]. In fact, mesenchymal or EMT CTCs have already been reported to be associated with disease progression and with the presence of distant metastasis in different malignancies [57,58]. Hence, the identification of CTCs according to their EMT phenotypes may provide valuable information in the clinical setting. ...
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Gastrointestinal (GI) cancers constitute a group of highest morbidity worldwide, with colorectal cancer (CRC) and gastric cancer being among the most frequently diagnosed. The majority of gastrointestinal cancer patients already present metastasis by the time of diagnosis, which is widely associated with cancer-related death. Accumulating evidence suggests that epithelial-to-mesenchymal transition (EMT) in cancer promotes circulating tumor cell (CTCs) formation, which ultimately drives metastasis development. These cells have emerged as a fundamental tool for cancer diagnosis and monitoring, as they reflect tumor heterogeneity and the clonal evolution of cancer in real-time. In particular, EMT phenotypes are commonly associated with therapy resistance. Thus, capturing these CTCs is expected to reveal important clinical information. However, currently available CTC isolation approaches are suboptimal and are often targeted to capture epithelial CTCs, leading to the loss of EMT or mesenchymal CTCs. Here, we describe size-based CTCs isolation using the RUBYchip™, a label-free microfluidic device, aiming to detect EMT biomarkers in CTCs from whole blood samples of GI cancer patients. We found that, for most cases, the mesenchymal phenotype was predominant, and in fact a considerable fraction of isolated CTCs did not express epithelial markers. The RUBYchip™ can overcome the limitations of label-dependent technologies and improve the identification of CTC subpopulations that may be related to different clinical outcomes.
... Pan-CK is the epithelial-specific tumor marker in liver cells and in the epithelium of bile ducts which is used for the identification of circulating tumor cells (56,57). Moreover, the tumor cells can undergo the epithelial to mesenchymal transition, which can result in downregulation of epithelial cell-specific molecules including cytokeratins (25), and this transition may grant tumor cells to stem cell properties enabling self-renewal (58). ...
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Citation: Magdy M.A., Omar The liver is the largest and most vulnerable organ in the body, several factors can cause liver cirrhosis and the onset of hepatocellular carcinoma. This study aims to investigate the possibility for erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) as adenosine deaminase inhibitor which reduces liver injury, neutrophil infiltration and the levels of proinflammatory cytokines and also it is considering as a target of liver cirrhosis and beginning of hepatocellular carcinoma protective, quercetin which is one of the most common flavonoids has an antioxidant, antitumor and chemopreventive effect on the liver-induced preneoplastic lesions and their combination against thioacetamide as a hepatotoxic and a carcinogenic compound. Biochemical, histopathological and immunohistochemical studies were carried out on male albino rats model to evaluate this possibility. Thioacetamide-treated rats showed a significant increase in liver functions tests (P = 8.6 X 10-7 for alanine transaminase, P = 1.89 X 10-8 for γ-glutamic transpeptidase and P = 8.8 X 10-13 for total bilirubin), α-fetoprotein (P = 7.95 X 10-13) and the number of Ki67+ cells (P = 3.0 X 10-6), and caused an elevation in the expression of inducible nitric oxide synthase (iNOS) and Pan-cytokeratin (Pan-CK) in hepatic tissue of rats.
... A larger percentage of patients (19/30; 63.3%) had > 50% of their CTCs double-positive for KLF8 and vimentin, whereas 36.7% (11/30) had 50% KLF8 þ /vimentin þ CTCs. Nel et al 53 showed that vimentin þ /CK þ ratio higher than 0.5, meaning that there were half as many vimentin positive cells as CK þ cells, was associated with a longer median time to progression (hazard ratio ¼ 0.18; 95% confidence interval: 0.01-2.75; P ¼ 0.03). ...
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Background Circulating tumor cells (CTCs) with an epithelial-mesenchymal transition phenotype in peripheral blood may be a useful marker of carcinomas with poor prognosis. The aim of this study was to determine the prognostic significance of CTCs expressing Krüppel-like factor 8 (KLF8) and vimentin in pancreatic cancer (PC). Methods CTCs were isolated by immunomagnetic separation from the peripheral blood of 40 PC patients before undergoing surgical resection. Immunocytochemistry was performed to identify KLF8 ⁺ and vimentin ⁺ CTCs. The associations between CTCs and time to recurrence (TTR), clinicopathologic factors, and survival were assessed. Univariate and multivariate analyzes were performed to identify risk factors. Results Patients with CTCs ( n = 30) had a higher relapse rate compared to those without ( n = 10) (70.0% vs 20.0%; P < 0.01). The proportion of KLF8 ⁺ /vimentin ⁺ CTCs to total CTCs was inversely related to TTR ( r = −0.646; P < 0.01); TTR was reduced in patients with > 50% of CTCs identified as KLF8 ⁺ /vimentin ⁺ ( P < 0.01). Independent risk factors for recurrence were perineural invasion and > 50% KLF8 ⁺ /vimentin ⁺ CTCs (both P < 0.05). Conclusion Poor prognosis can be predicted in PC patients when > 50% of CTCs are positive for KLF8 and vimentin.
... In our preliminary analysis, K16 expression was associated with higher tumor aggressiveness, and it was mainly associated with epithelial-mesenchymal plasticity. Indeed, a significant relationship has been reported in several studies between the CTCs with high epithelial-mesenchymal plasticity and poor clinical outcomes in different cancer entities [5,[42][43][44][45][46]. These CTCs have dynamic plasticity to adapt to the selective microenvironment during their dissemination in distant organs [5,43]. ...
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... In our study, we relied on the expression of the epithelial adhesion molecule (EpCAM) for the detection of CTCs. EpCAM is a well-characterized surface marker of liver cancer cells, and the presence of EpCAM-positive CTCs has been previously associated with vascular invasion, increased serum AFP [21,34,35], more advanced HCC stage, and increased recurrence rates after surgery [21,36]. There are several approaches to enumerate EpCAMpositive CTCs, the most widely known being the CellSearch TM system, which was used in some preliminary studies including HCC patients who underwent surgical resection of HCC [19,30,33]. ...
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... Up to now various EpCAM-independent approaches like density gradient centrifugation [7], microfiltration dependent on size and deformability [8], other 2Dand 3D membrane microfilters [9,10], microfluidic approaches using bioelectric properties [11] and several CTC chambers, -channels and -chips [12][13][14] commonly based on distinct cellular and biophysical differences between CTCs and blood cells are available [15][16][17][18]. A promising method is the so-called negative CTC selection by depletion of hematopoietic cells using magnetically labeled antibodies against CD45 to enrich non-hematopoietic cells in the remaining cell suspension [19,20]. These non-hematopoietic cells include possible CTC candidates with not only epithelial but also mesenchymal-or stemcell-like characteristics. ...
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... The increase in the percentage of CTCs positive for the mesenchymal marker is higher in metastatic breast cancer than in early-stage breast cancer [57], denoting a more aggressive and metastatic potential of mesenchymal CTCs than those with epithelial markers alone [56,73,76,[78][79][80]101,102]. Hybrid CTCs, cytokeratins + /vimentin + /CD45 − , are also associated with disease progression and the presence of distant metastasis [103,104]. EMT-CTCs have specific markers that allow a more accurate prediction of worse prognosis and metastasis. BCAT1-induced EMT-CTCs may be an important biomarker of hepatocellular carcinoma metastasis [105]. ...
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