Download Circulating stem cell–like epithelial cell adhesion molecule–positive

Circulating Stem Cell–Like Epithelial Cell Adhesion
Molecule–Positive Tumor Cells Indicate Poor
Prognosis of Hepatocellular Carcinoma After Curative
Resection
Yun-Fan Sun,1* Yang Xu,1* Xin-Rong Yang,1* Wei Guo,1,2 Xin Zhang,1 Shuang-Jian Qiu,1 Ruo-Yu Shi,1
Bo Hu,1 Jian Zhou,1,3 and Jia Fan1,3
Epithelial cell adhesion molecule–positive (EpCAM1) hepatocellular carcinoma (HCC)
cells may constitute a tumor-initiating subpopulation in tumorigenic cell lines and HCC
specimens. In the present study, EpCAM1 circulating tumor cells (CTCs) were identified
prospectively in HCC patients undergoing curative resection, and the prognostic significance and their stem cell–like characteristics were investigated further. Blood samples
from 123 HCC patients were tested prior to resection and 1 month thereafter. CTCs were
present in 66.67% of patients, and the cell count measured in 7.5 mL of blood (CTC7.5)
ranged between 1 and 34. Fifty-one patients had CTC7.5 of 2 preoperatively, and these
patients developed tumor recurrence earlier than those with CTC7.5 of <2 CTCs (P <
0.001). A preoperative CTC7.5 of 2 was an independent prognostic factor for tumor recurrence (P < 0.001). Its prognostic significance also applied to patients with alpha-fetoprotein (AFP) levels of 400 ng/mL or subgroups with low recurrence risk (all P < 0.05).
A significant decrease of CTC-positive rates (66.67% to 28.15%, P < 0.05) and CTC7.5
values (2.60 6 0.43 to 1.00 6 0.36, P < 0.05) was observed 1 month after resection.
Patients with consistent CTC7.5 <2 had lower recurrence rates than those with values consistently 2 (15.5% versus 87.50%, P < 0.001). EpCAM1 CTCs displayed cancer stem
cell biomarkers (CD133 and ABCG2), epithelial-mesenchymal transition, Wnt pathway
activation, high tumorigenic potential, and low apoptotic propensity. Conclusion: Stem
cell–like phenotypes are observed in EpCAM1 CTCs, and a preoperative CTC7.5 of 2 is a
novel predictor for tumor recurrence in HCC patients after surgery, especially in patient
subgroups with AFP levels of 400 ng/mL or low tumor recurrence risk. EpCAM1 CTCs
may serve as a real-time parameter for monitoring treatment response and a therapeutic
target in HCC recurrence. (HEPATOLOGY 2013;57:1458-1468)
H
epatocellular carcinoma (HCC) is one of the
most prevalent malignancies worldwide, and
associated morbidity and mortality rates have
escalated in recent years.1 Despite improvements in
surveillance and clinical treatment strategies, the prognosis of HCC remains very poor due to high incidence
of recurrence and metastasis.2 Traditional clinicopathological parameters such as tumor morphology,
Abbreviations: AFP, alpha-fetoprotein; AUC, area under the curve; BCLC, Barcelona Clinical Liver Cancer; CI, confidence interval; CK, cytokeratin; CSC,
cancer stem cell; CTC, circulating tumor cell; DAPI, 4’,6-diamidino-2-phenylindole; EpCAMþ, epithelial cell adhesion molecule–positive; HCC, hepatocellular
carcinoma; mRNA, messenger RNA; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; qRT-PCR, quantitative real-time polymerase chain
reaction; ROC, receiver operating characteristic; TACE, transcatheter arterial chemoembolization; TTR, time to recurrence.
From the 1Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, and Key Laboratory of Carcinogenesis and Cancer Invasion,
Ministry of Education, Shanghai 200032, P. R. China; 2Department of Laboratory Medicine, Zhongshan Hospital, Fudan University; 3Institute of Biomedical Sciences,
Fudan University, Shanghai 200032, P. R. China
Received March 12, 2012; accepted October 27, 2012.
Supported by grants from the Major Program of National Natural Science Foundation of China (81030038), the National Key Sci-Tech Project
(2012ZX10002011-002, 2013ZX10002011-004, 2012ZX0930100-007, and 2012ZX10002-016), the National Natural Science Foundation of China
(81000927, 81071661, 81172277, and 30972949), the Research Fund for the Doctoral Program of Higher Education of China (20100071120064), and the
Shanghai New Project for Excellent Youth (XYQ2011020).
National Science Foundation for Distinguished Young Scholars of China (81225019); Shanghai Rising-Star Follow-up Program Fundings (10QH1400500).
*These authors contributed equally to this work.
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HEPATOLOGY, Vol. 57, No. 4, 2013
histopathological features, and tumor staging system
offer limited information for predicting postoperative
recurrence and fail to monitor the therapeutic response
in a real-time manner.3 Therefore, it is imperative to
develop novel approaches for discriminating high-risk
factors of recurrent patients and continuous surveillance of antitumor treatment response.
The spread of circulating tumor cells (CTCs) in the
blood plays a major role in the initiation of metastases
and tumor recurrence after surgery.3 Recent studies
have reported that stem cell markers are frequently
overexpressed in CTCs of metastatic breast cancer.4 In
addition, clinical observations and animal model studies indicate that although thousands of tumor cells disseminate into the circulation, only a small population
with stem cell–like properties survives migration to establish secondary colonies.5,6 Therefore, CTCs with
stem cell properties might be potential sources for cancer relapse and distant metastasis, consistent with the
cancer stem cell (CSC) hypothesis.7
The US Food and Drug Administration–approved
CellSearch system, which enriches CTCs via anti-EpCAM
antibody conjugated to ferrofluid magnetic particles, has
been reported to enumerate CTCs in some specific types
of malignancies.8 However, there has been no report of
applying this method to detection of CTCs in HCC
patients, and the prognostic and biological relevance of
EpCAMþ CTCs in HCC patients remains unclear. In
our previous work, we confirmed that EpCAMþ HCC
cells derived from cell lines and tumor specimens were
highly invasive and tumorigenic, and EpCAM could serve
as a biomarker for tumor-initiating cells in HCC.9,10
Thus, detection of CTCs by EpCAM expression may
indicate the more aggressive stem cell–like CTCs in
HCC. Further identification of biological characteristics
of this CTC subpopulation could lead to development of
novel targeted drugs and extract more information on the
mechanisms of metastasis in this cancer.
In this study, we hypothesized that EpCAMþ CTCs
embed CSC properties and were one of the potential
sources of HCC recurrence and metastasis, and therefore
their detection might correlate with an adverse clinical
outcome. To test the hypothesis, we used a standardized
CellSearch method to prospectively explore the prevalence, dynamic changes, and prognostic significance of
SUN, XU, YANG, ET AL.
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these cells in HCC patients undergoing curative resection. In addition, expression of CSC-related molecules,
apoptotic propensity, and tumorigenic capacity were
investigated in EpCAMþ CTCs.
Patients and Methods
Patients and Specimens. From July 2010 to June
2011, 123 HCC patients undergoing curative resection
were recruited into a prospective study. The entrance
criteria were: (1) definitive pathological diagnosis of
HCC based on World Health Organization criteria;
(2) curative resection, defined as complete macroscopic
removal of the tumor11; and (3) no prior anticancer
treatment. Tumor stage was determined according to
the Barcelona Clinic Liver Cancer (BCLC) staging system,12 and tumor differentiation was defined according
to the Edmondson grading system.13 In addition, 10
healthy donors and five patients with benign liver disease were enrolled as negative controls.
The time points for blood collection were 2 days before
resection (baseline), and a median of 31 days (range, 2748 days) after resection. Samples of 7.5 mL were collected
and used for CellSearch analysis. A second blood sample
(7.5 mL) for confocal microscopic analysis was obtained
prior to surgery from the 82 patients who were positive
for preoperative EpCAMþ CTCs. Additional samples
were taken from selected individuals for use in quantitative
real-time polymerase chain reaction (qRT-PCR) assays (30
HCC patients and 20 healthy volunteers, 10 mL blood
per patient) and tumorigenic assays (six HCC patients, 30
mL blood per patient). Ethical approval for the use of
human subjects was obtained from the Research Ethics
Committee of Zhongshan Hospital consistent with ethical
guidelines of the 1975 Declaration of Helsinki, and
informed consent was obtained from each patient.
Follow-up and Tumor Recurrence. Postoperative
patient surveillance was performed as described.14 A diagnosis of recurrence was based on computed tomography scans, magnetic resonance imaging, digital subtraction angiography and raised serum alpha-fetoprotein
(AFP) level, with or without histological confirmation.
Follow-up was terminated on June 30, 2012. Time to
recurrence (TTR) was defined as the interval between
resection and the diagnosis of any type of recurrence,15
Address reprint requests to: Jia Fan, M.D., Ph.D., Liver Cancer Institute, Zhongshan Hospital, Fudan University, 136 Yi Xue Yuan Road, Shanghai 200032,
People s Republic of China. E-mail: [email protected]; fax: (86)-21-64037181; or Jian Zhou, M.D., Ph.D., Liver Cancer Institute, Zhongshan Hospital,
Fudan University, 136 Yi Xue Yuan Road, Shanghai 200032, People s Republic of China. E-mail: [email protected]; fax: (86)-21-64037181.
C 2013 by the American Association for the Study of Liver Diseases.
Copyright V
View this article online at wileyonlinelibrary.com.
DOI 10.1002/hep.26151
Potential conflict of interest: Nothing to report.
Additional Supporting Information may be found in the online version of this article.
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with intrahepatic recurrence and extrahepatic metastasis
defined as the end points.16 We defined recurrence
within 1 year after surgery as early recurrence.17
CD45 Depletion, Messenger RNA Isolation,
Reverse-Transcription, and qRT-PCR. Cells were
enriched from blood samples within 8 hours after collection using the RosetteSep Human CD45 Depletion
Cocktail (StemCell Technologies, Vancouver, Canada) as
described.18 The CD45-depleted fraction was subjected to
messenger RNA (mRNA) isolation using the RNeasy
Micro Kit (QIAGEN, Valencia, CA). Subsequently,
reverse transcription was performed using the Quantitect
Reverse Transcription Kit (Qiagen). Analysis by qRT-PCR
was done with the Light Cycler 480 system (Roche Diagnostics, Basel, Switzerland). All procedures were performed according to the manufacturer’s instructions.
Gene expression levels were calculated according to the
following equation: 2DCT [DCT ¼ Ct(target) Ct(bactin)]. PCR conditions were as follows: 10 minutes at
95 C, followed by 45 cycles of 95 C for 10 seconds and
60 C for 60 seconds. Every sample was measured in triplicate. The primers used are listed in Supporting Table 1.
Enumeration of CTCs. EpCAMþ CTC analysis was
performed using CellSearch (Veridex, Raritan, NJ) as
described,19 without knowledge of patient clinical characteristics. Results of CTC enumeration were expressed
as the number of cells per 7.5 mL of blood (CTC7.5).
Analysis of EpCAM1 CTCs via Confocal
Microscopy. Blood samples were processed using the
CellSearch Profile kit (Veridex) to isolate and collect
EpCAMþ cells,20 and cells in the isolated fraction were
prepared by cytospin (Thermo Fisher, Waltham, MA) and
subjected to immunofluorescence analysis as described.20
The antibodies used in the study are listed in Supporting
Table 2. All samples were analyzed with a Zeiss confocal
microscope (Carl Zeiss, Oberkochen, Germany).
Isolation of Circulating EpCAM1/CD452 Cells
and Tumorigenic Assay. To ensure that enough
EpCAMþ CTCs were harvested for tumorigenic assay,
we collected 30 mL blood from each of the six
patients who had advanced HCC with portal vein
thrombosis. Mononuclear cells from whole blood were
isolated by density gradient centrifugation using FicollPaque PLUS medium (GE Healthcare,Waukesha, WI)
within 1 hour after collection. The isolated cells were
then subjected to magnetic-activated cell sorting (Milteny Biotec GmbH, Bergisch Gladbach, Germany), to
purify EpCAMþ/CD45 CTCs by CD45 depletion
and EpCAM selection. All procedures were performed
according to the manufacturer’s instructions.
Four-week-old nonobese diabetic/severe combined
immunodeficiency (NOD/SCID) mice were purchased
HEPATOLOGY, April 2013
from the Shanghai Laboratory Animal Commission of
the Chinese Academy of Science, Shanghai, China.
Cells to be tested were suspended in 100 lL of Dulbecco’s modified Eagle’s medium and Matrigel (1:1).
Approximately 300 EpCAMþCD45 or 1 104
EpCAMCD45 cells were injected subcutaneously
into the flank of each NOD/SCID mice. The size and
incidence of subcutaneous tumors were recorded every
week. These procedures were approved by The Animal
Care and Use Committee of Fudan University.
Statistical Analysis. The cutoff value used in prognosis was estimated using X-tile 3.6.1 software (Yale
University, New Haven, CT).21 The results indicated
that in blood, a threshold CTC7.5 value of 2 showed
the most significant power to predict patient outcome
(Supporting Fig. 1); therefore, it was used in all further
analyses. Receiver operating characteristic (ROC) analysis confirmed that this level was the optimal cutoff.
Statistical analyses were performed with SPSS version 19.0 for Windows (IBM). Data are presented as
the mean 6 SEM. A chi-squared test, Fisher’s exact
test, and Student t test were used for comparison
between groups where appropriate. The relationship
between the TTR and CTC counts was analyzed using
Kaplan-Meier survival curves and a log-rank test. Univariate and multivariate analyses were based on the
Cox proportional hazard regression model. P < 0.05
was considered statistically significant. ROC curve
analysis was used to determine the predictive value of
the parameters, and the differences in the area under
the curve (AUC) were detected using Stata version 10
(StataCorp, College Station, TX).
Results
High Expression Levels of EpCAM mRNA in
CD45-Depleted Peripheral Blood Mononuclear
Cells. The mRNA levels of four putative hepatic CSC
biomarkers (EpCAM, CD133, CD90, and ABCG2)
were determined via qRT-PCR analysis in CD45depleted peripheral blood mononuclear cells of 30
HCC patients and 20 healthy volunteers. The expression of EpCAM was significantly higher in cells of
HCC patients versus healthy controls (P < 0.05),
whereas there was no significant difference in the
expression of CD133, CD90, or ABCG2 between the
groups (P > 0.05) (Fig. 1A). These data suggested
that EpCAM might be a reliable biomarker to identify
circulating CSCs in HCC.
Detection of EpCAM1 CTCs. Because the mRNA
level of EpCAM was highly expressed in CD45depleted peripheral blood mononuclear cells of HCC
HEPATOLOGY, Vol. 57, No. 4, 2013
SUN, XU, YANG, ET AL.
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Fig. 1. Identification and some CSC characteristics of EpCAMþ CTCs. (A) mRNA expression levels of four putative cancer stem cell markers in
CD45-depleted blood samples of healthy volunteers (n ¼ 20) and HCC patients (n ¼ 30). (B) Typical images of EpCAMþ CTCs identified with
the CellSearch system. CTCs are defined as cells staining positively for CK and DAPI and negatively for CD45. Circulating tumor microemboli
(CTM) are defined as CTC clusters. Apoptotic CTCs are defined as CTCs with fragmented, condensed DAPI-stained nuclei morphology. (C) Expression of CSCs and epithelial-mesenchymal transition–related markers in EpCAMþ CTCs. (D) Representative NOD/SCID mice tested for tumorigenicity of subcutaneously injected EpCAMþ/CD45 or EpCAM/CD45 CTCs (upper panels) and hematoxylin and eosin (H&E) staining of
subcutaneous nodules (lower panels). Scale bar, 100 lm.
patients, we investigated the prevalence of EpCAMþ
CTCs in HCC patients using the CellSearch system.
CTCs detected with the CellSearch system were
defined as nucleated intact cells that were positive for
cytokeratins and negative for CD45 (Fig. 1B).8 Apoptotic CTCs, defined as CTCs with fragmented, condensed 4’,6-diamidino-2-phenylindole (DAPI)-stained
nuclear,22 were also enumerated and examples were
shown in Fig. 1B. The apoptotic cells were excluded
from the CTC counts and recorded separately.
Preoperatively, EpCAMþ CTCs were detected in 82
of 123 HCC patients at CTC7.5 levels within a range
of 1-34, and 51 patients had counts of 2. No CTCs
were detected in 41 HCC patients or in any of the
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HEPATOLOGY, April 2013
Table 1. Expression Profile of Stem Cell–Like and EpithelialMesenchymal Transition Markers in Nucleated EpCAM1/
CK1/CD452 CTCs From HCC Patients Positive for EpCAM1
CTCs
Markers
CD133
ABCG2
CD90
b-Catenin
Vimentin/E-cadherin
Vimentinþ/E-cadherin
Vimentin/E-cadherinþ
Vimentinþ/E-cadherinþ
Patients with
Marker-Positive CTCs
14/17
12/16
0/16
10/17
(82.35)
(75.00)
(0.00)
(58.82)
10/16 (62.5)
3/16 (18.75)
4/16 (25.00)
Marker-Positive
CTCs
54/65
43/59
0/51
37/69
(83.07)
(72.88)
(0.00)
(53.62)
38/59 (64.41)
6/59 (10.17)
8/59 (13.56)
Columns on the right represent the number of patients with at least one CTC
that stains positive for a given marker and number of CTCs scoring positive for
each marker. Data are presented as no. (%).
blood samples derived from healthy volunteers or
patients with benign liver disease.
Stem Cell–Like Phenotypes Found in EpCAM1
CTCs. Eighty-two blood samples from patients with
positive preoperative EpCAMþ CTCs were processed
using the CellSearch Profile procedure and divided
into five triple-staining panels according to random
numbers generated by SPSS, including panel 1 for
CD133/cytokeratin (CK)/CD45 (n ¼ 17), panel 2 for
ABCG2/CK/CD45 (n ¼ 16), panel 3 for CD90/CK/
CD45 (n ¼ 16), panel 4 for b-catenin/CK/CD45 (n
¼ 17), and panel 5 for vimentin/E-cadherin/CD45 (n
¼ 16) (Fig. 1C, Supporting Fig. 2, and Table 1).
CD133þ/CKþ/CD45 cells were identified in 14 of
17 patients, and accounted for 54 of 65 EpCAMþ
CTCs from these patients. Twelve of 16 patients had
ABCG2þ/CKþ/CD45 cells, and ABCG2 was
expressed in 43 of 59 detectable CTCs. None of 16
patients had detectable CD90þ/CKþ/CD45 cells
(Supporting Fig. 2). We found evidence of cytoplasmic/nuclear b-catenin accumulation of EpCAMþ
CTCs in 10 of 17 patients, and 37 of 69 cells
observed showed b-catenin accumulation (cytoplasmic
versus nuclear: 37 versus 15). Among 16 patients
staining for vimentin/E-cadherin/CD45, three patients
had vimentin/E-cadherinþ/CD45 CTCs, 10 had
vimentinþ/E-cadherin/CD45 cells, and four coexpressed vimentin and E-cadherin on CD45 cells.
Vimentin/E-cadherinþ/CD45,
vimentinþ/Ecadherin/CD45,
and
vimentinþ/E-cadherinþ/
CD45 cells accounted for 6, 38, and 8 EpCAMþ
CTCs, respectively, of the total of 59 observed. The
details are shown in Table 1.
EpCAM1 CTCs in HCC Patients Might Have a
Survival Advantage. Apoptotic status of CTCs was
investigated in the cohort of 123 HCC patients. CTCs
with apoptotic morphology were observed in 19 of 82
patients who had positive preoperative CTCs, and apoptotic cells accounted for 29 of 348 overall EpCAMþ
CTCs (nonapoptotic and apoptotic) that were examined. The apoptotic ratio of total EpCAMþ CTCs in
HCC (8.3%) in our study is lower than reported for
other tumor types (20%-54%).22,23
Table 2. Clinical Characteristics of HCC Patients and
Correlation With EpCAM1 CTC Counts
Clinical
Characteristics
Age, years
50
>50
Sex
Male
Female
HBsAg
Negative
Positive
Child-Pugh score
A
B
Liver cirrhosis
No
Yes
ALT, U/L
75
>75
AFP, ng/mL
400
>400
No. of tumors
Single
Multiple
Tumor size, cm
5
>5
Tumor encapsulation
Complete
None
Satellite lesion
No
Yes
Vascular invasion
No
Yes
Edmondson stage
I-II
II-IV
BCLC stage
0þA
BþC
Recurrence
No
Yes
No. of Patients
(N ¼ 123)
CTC7.5 <2
CTC7.5 2
64
59
39
33
25
26
115
8
69
3
46
5
12
111
9
63
3
48
122
1
71
1
51
0
29
94
17
55
12
39
102
21
63
9
39
12
70
53
48
24
22
29
112
11
65
7
47
4
80
43
51
21
29
22
77
46
22
50
24
27
106
17
68
4
38
13
78
45
56
16
22
29
83
40
57
15
26
25
101
22
61
11
40
11
72
51
57
15
15
36
P
0.573
0.212*
0.223*
0.398†
0.992
0.109
0.009
0.719*
0.109
0.062
0.002
<0.001
0.001
0.370
<0.001
Abbreviations: ALT, alanine aminotransferase; HBsAg, hepatitis B surface
antigen.
*Continuous correction.
†Fisher’s exact test.
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SUN, XU, YANG, ET AL.
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Fig. 2. EpCAMþ CTCs correlate significantly with early recurrence. (A) Distribution of CTCs in early recurrent patients (!), nonrecurrent
patients (), healthy volunteers (~), and benign liver disease patients (*) (P ¼ 0.006). (B) Kaplan-Meier analysis for time to recurrence in
HCC patients with CTC7.5 <2 or 2 preoperatively. (C) Predictive ability of CTC7.5 2 was compared with other clinical parameters by ROC
curves in 123 HCC patients. The AUC with 95% CI for time to recurrence are also shown. *P < 0.05 versus CTC7.5 2.
EpCAM1 CTCs Exhibited Tumorigenicity Properties. A total of 10 NOD/SCID mice were used in
tumorigenicity transplantation experiments, with
EpCAMþ/CD45 cells being tested in six mice, and
EpCAM/CD45 in four mice. Three months after
injection, 50% of the mice injected with
EpCAMþCD45 cells developed subcutaneous nodules, while none developed from EpCAMCD45
cells (Fig. 1D). The weights and sizes of subcutaneous
tumor nodules are shown in Supporting Fig. 3.
Prevalence of EpCAM1 CTC Counts Before Resective Surgery and Their Prognostic Significance. Patient demographics are listed in Table 2. By the time
of analysis, early recurrence had occurred in 51 of 123
patients, with a mean follow-up time of 15.1 6 2.3
months (median, 14.6 months; range, 12.3-23.2
months). Among these 51 patients, 39 had intrahepatic recurrence only, five had lung metastasis only,
and seven suffered both intrahepatic recurrence and
lung metastasis.
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HEPATOLOGY, April 2013
Table 3. Univariate and Multivariate Cox Proportional Regression Analysis of Factors Associated With Recurrence
Univariate Analysis
Multivariate Analysis
Variables
HR (95% CI)
P
HR (95% CI)
P
Age, >50 years versus 50 years
Sex, male versus female
HBsAg, positive versus negative
Liver cirrhosis, yes versus no
Child-Pugh score, B versus A
ALT, >75 U/L versus 75 U/L
AFP, >400 ng/mL versus 400 ng/mL
No. of tumors, multiple versus single
Tumor size, >5 cm versus 5 cm
Tumor encapsulation, none versus Complete
Satellite lesion, yes versus no
Vascular invasion, yes versus no
Edmondson stage, III-IV versus I-II
BCLC stage, BþC versus 0þA
Preoperative CTC7.5, 2 versus <2
0.97 (0.56-1.68)
2.02 (0.80-5.10)
3.38 (0.82-13.89)
0.95 (0.50-1.78)
0.05 (0-8,631.53)
1.22 (0.61-2.43)
1.90 (1.09-3.30)
1.07 (0.43-2.70)
2.61 (1.50-4.53)
2.13 (1.23-3.68)
4.01 (2.15-7.72)
2.53 (1.46-4.40)
1.70 (0.97-2.97)
2.39 (1.61-4.37)
5.37 (2.92-9.85)
0.916
0.135
0.092
0.863
0.625
0.577
0.023
0.881
0.001
0.007
<0.001
0.001
0.063
0.005
<0.001
NA
NA
NA
NA
NA
NA
1.15 (0.63-2.05)
NA
2.36 (1.28-4.32)
1.82 (1.01-3.26)
2.70 (1.34-5.44)
0.89 (0.47-1.71)
NA
NA
5.20 (2.65-10.21)
NA
NA
NA
NA
NA
NA
0.642
NA
0.006
0.045
0.005
0.733
NA
NA
<0.001
Clinicopathological variables were adopted for their prognostic significance by univariate analyses.
Abbreviations: ALT, alanine aminotransferase; HBsAg, hepatitis B surface antigen; HR, hazard ratio; NA, not applicable.
CTC counts were significantly higher in recurrent
patients than in nonrecurrent ones (mean, 4.1 versus
1.5, respectively; P ¼ 0.006) (Fig. 2A). Recurrence
was observed in 36 of 51 patients with preoperative
CTC7.5 of 2, whereas only 15 of 72 patients with
values of <2 recurred. Among 12 patients who developed lung metastasis, eight patients had CTC7.5 of 2
prior to resection. In addition, patients with preoperative values of 2 were more likely to have satellite
lesions (P ¼ 0.002), vascular invasion (P < 0.001),
poor tumor differentiation (P ¼ 0.001), and higher serum AFP level (P ¼ 0.009) (Table 2).
Using a CTC7.5 of 2 as the cutoff value in univariate
analysis, preoperative CTC7.5 counts showed prognostic
significance for TTR (P < 0.001) (Table 3). Patients
with counts 2 had significantly shorter TTR (median,
4.9 months versus not reached) and higher recurrence
rates (70.6% versus 20.8%) than those with CTC7.5 of
<2 (P < 0.001) (Fig. 2B). Levels of AFP, tumor size,
tumor encapsulation, satellite lesion, vascular invasion,
and BCLC stage were also unfavorable prognostic variables for recurrence (P < 0.05) (Table 3). Because
BCLC stage was associated with the three clinical categories of tumor characteristics, liver function and performance status, it was not included in multiple analyses to avoid potential bias. In multivariate analysis, a
CTC7.5 of 2 was the strongest independent prognostic
factor for TTR (hazard ratio, 5.20; 95% confidence
interval [CI], 2.65-10.21; P < 0.001) (Table 3).
The AUC for a CTC7.5 of 2 was 0.750, with a sensitivity of 70.60% and specificity of 80.00% (P <
0.001; 95% CI, 0.66-0.84). Compared with other
clinical indices, a CTC7.5 of 2 prior to resection was
the strongest factor for predicting early recurrence in
HCC (AUCs with 95% CI for TTR; P < 0.05 versus
CTC 2) (Fig. 2C).
The prognostic significance of preoperative CTC7.5
within clinical subgroups was further investigated. In
patients with AFP 400 ng/mL, we found that patients
with a CTC7.5 of 2 had higher recurrence rates
(68.20% versus 8.33%) and shorter TTR (median, 5.0
months versus not reached) than those with <2 (P <
0.001) (Fig. 3A). Patients with preoperative CTC7.5 of
2 showed a relatively higher risk of developing postoperative recurrence than those with <2 in low recurrence risk subgroups, including tumor size 5 cm
(62.07% versus 13.73%; P ¼ 0.001), single tumor
(68.09% versus 21.54%; P < 0.001), absence of satellite lesions (63.16% versus 20.59%; P < 0.001), absence of vascular invasion (68.18% versus 16.07%; P <
0.001), Edmondson stage I-II (73.07% versus 19.30%;
P < 0.001), and BCLC stage 0þA (67.50 % versus
14.75%; P < 0.001) (Figs. 3B-H).17,24
The Dynamic Changes of EpCAM1 CTC Counts
After Surgery and Their Prognostic Significance. The
postoperative levels were measured in 103 patients at 1
month following resection. Both the CTC-positive
rates (66.67% to 28.15%; P < 0.05) and CTC7.5 values (2.60 6 0.43 to 1.00 6 0.36; P < 0.05) dropped
dramatically after surgery (Fig. 4A).
Based on changes between preoperative and postoperative CTC7.5, 103 patients were divided into four
groups: I, persistent levels of 2 CTCs (n ¼ 8) at the
two time points; II, preoperatively 2 then postoperatively <2 (n ¼ 31); III, preoperatively <2 then postoperatively 2 (n ¼ 6); and IV, persistent <2 (n ¼ 58).
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Fig. 3. Kaplan-Meier analysis of EpCAMþ CTCs in subgroups of HCC patients. The prognostic value of EpCAMþ CTCs was significant in (A) the
AFP 400 ng/mL and (B-H) clinical low recurrence risk subgroups, including (B) tumor size 5 cm, (C) single tumor, (D) no satellite lesions, (E) no
vascular invasion, (F) Edmondson stage I-II, and (G) BCLC stage 0þA. (H) Recurrent rates of patients with CTC7.5 <2 versus 2 CTCs in the subgroups for tumor size 5 cm, single tumor, no satellite lesion, no vascular invasion, Edmondson stage I-II, BCLC stage 0þA, and AFP 400 ng/mL.
The recurrence rates for groups I-IV were 87.50%,
61.3%, 66.7%, and 15.5%, respectively. Patients in
group I showed significantly shorter TTR and higher
recurrence rates than group IV (median TTR of 2.2
versus not reached; recurrence of 87.5% versus 15.5%;
P < 0.001) and a propensity of increased recurrence
compared with group II (median TTR, 2.2 months versus not reached; recurrence, 87.50% versus 61.3%; P ¼
0.073) and while there was no significant difference
among recurrence rates of groups I and III (P ¼ 0.241)
(Fig. 4B). Compared with group IV, patients in the
other three groups had significantly shorter TTR and
higher recurrence rates (P < 0.001) (Fig. 4B).
Discussion
The most effective therapeutic options for HCC
offering a favorable prognosis are hepatectomy and
liver transplantation. However, even such presumably
curative surgery does not guarantee full recovery, and
1466
SUN, XU, YANG, ET AL.
HEPATOLOGY, April 2013
Fig. 4. Changes in EpCAMþ CTC load reflect treatment response to HCC resection. (A) Changes in CTC load before versus after surgery. (B)
Prognostic significance of CTC load for time to recurrence in patients with persistent CTC7.5 2, conversion of CTC7.5 from 2 to <2, conversion
of CTC7.5 from <2 to 2, and persistent CTC7.5 of <2.
this failure is due in large part to the high incidence of
recurrence (50%-70% at 5 years).2 The most significant reason for the unsatisfactory therapeutic outcome
is residual micrometastases formed prior to resection
or dissemination of tumor cells during surgical manipulation.25 Unfortunately, routine diagnostic approaches
are thus far unable to identify the HCC patient subpopulation at high risk of developing micrometastases
preoperatively,17 as well as the tumor cells that escape
or invade into peripheral blood during surgery. Recent
clinical studies have provided evidence that CTCs may
directly participate in the metastasis cascade in various
types of malignancies.26 The prognostic significance of
CTCs has been widely reported in metastatic breast,
colon, and prostate cancers. However, the presence of
CTCs in the circulation is a necessary but insufficient
condition for the initiation of metastasis, since only a
minority of dispersed cells possessing stem cell–like
properties is capable of reseeding the tissue of origin
or metastasizing to distant organs.3,6 Therefore, identifying the stem cell–like CTC subset with such properties would provide more clinically relevant prognostic
information than total CTC counts.
In the present study, we found that patients with
preoperative CTC7.5 levels of 2 EpCAMþ CTCs suffered significantly earlier recurrence (within 1 year)
than patients with lower levels. A preoperative
EpCAMþ CTC7.5 2 was significantly associated with
aggressive HCC phenotypes. Moreover, EpCAMþ
CTCs displayed stem cell–like traits. Based on these
data, we inferred that EpCAMþ CTCs with stem cell–
like phenotypes might represent a more aggressive subset of CTCs. These cells were more likely to invade
the circulatory system, survive, and finally seed in
orthotopic or distant sites, leading to local recurrence
or distant metastasis. Thus, the preoperative detection
of EpCAMþ CTCs might serve as a novel indicator
reflecting the micrometastatic status and recurrence
risk of HCC patients in a real-time manner, which in
turn could provide a therapeutic window and target
before the appearance of bona fide recurrence.
According to the CSC hypothesis, a small population of cells possessing stem cell–like traits is the driving force of tumor progression and resistance to classical therapies. Their presence in tumor tissues was
reported to be associated with a very poor clinical outcome in HCC patients.16,27 Whether CTCs from
HCC embed stem cell–like characteristics still requires
additional study. Here, we have found that EpCAMþ
CTCs preferentially coexpress CSC biomarkers, such
as CD133 and ABCG2, or exhibit cytoplasmic and
nuclear accumulation of b-catenin, which indicates
Wnt pathway activation.28 We also observed that
EpCAMþ CTCs in most patients displayed a mesenchymal phenotype with vimentinþ/E-cadherin (Fig.
1B), which is also an important property of CSCs.29
The apoptotic ratio of total EpCAMþ CTCs in HCC
(8.3%) in our study was lower than reported in other
tumor types (20%-54%).22,23 In addition, we also
observed that EpCAMþ/CD45 CTCs had high
HEPATOLOGY, Vol. 57, No. 4, 2013
tumorigenic potential, while EpCAM/CD45 cells
did not. All of these data indicated that EpCAMþ
CTCs in HCC embedded properties of cancer stem–
like cells, which might be the ‘‘seeds’’ of tumor metastasis and recurrence.7
In clinical practice, it is challenging to predict tumor relapse in low recurrence risk HCC subgroups.17,24 The present study is the first to show that
preoperative EpCAMþ CTC levels retain their prognostic value in those subgroups at risk for which conventional clinicopathological variables offer limited information predicting tumor recurrence. So far, AFP
level is the most extensively used diagnostic biomarker
and tumor recurrence indicator of HCC in AFP-positive patients.30 Clinical data demonstrated that low serum AFP concentration (e.g., 400 ng/mL) was associated with better clinical outcome. Nevertheless, it is
difficult to monitor recurrence in the 30%-40% of
HCC patients with low AFP levels.17,31 Here, we have
shown that determination of preoperative EpCAMþ
CTC level is a promising and feasible tool for recurrence prediction in patients with low AFP concentration. Large cohort studies should be undertaken to further validate the prognostic significance in this specific
HCC patient subpopulation.
The clinical use of monitoring CTC changes with
treatment has been reported in various types of cancers.32,33 However, the influence of surgical resection
of the primary tumor on CTC status in HCC remains
to be elucidated. In the present study, we report for
the first time that a significant decrease of CTC load
was observed soon after resection, which may well be
attributed to surgical resection of the primary tumor.
Patients whose CTC7.5 failed to drop to <2 postoperatively showed a propensity of increased recurrence,
and this suggested that CTC detection might be a surrogate indicator for surveillance of the response to the
HCC curative resection. Furthermore, in BCLC 0þA
patients, those who experienced a drop of CTC7.5 to
<2 postoperatively showed lower recurrence risk than
those with persistent levels of 2 CTCs (P ¼ 0.044;
data not shown). We propose that treatment response
surveillance by determination of CTC load may provide a powerful test enabling accurate and early decision making to tailor the most effective therapy
according to characteristics of individual tumors.
Our previous study indicated that postoperative adjuvant transcatheter arterial chemoembolization
(TACE) could improve the survival of patients with
risk factors for residual tumor.34 In our study, patients
with a high risk of recurrence, evidenced by clinical
features such as vascular invasion and microsatellite
SUN, XU, YANG, ET AL.
1467
lesions, were given one to three courses of prophylactic
TACE (doxorubicin, cisplatin, 5-fluorouracil, and
iodized oil) 1 month after surgery.35 We retrospectively
collected the data of HCC patients with 2 CTCs
who performed the prophylactic TACE and compared
the antirecurrence results with those who did not perform TACE, and found that prophylactic TACE was
beneficial in preventing recurrence in patients with 2
CTCs (P ¼ 0.006) (Supporting Fig. 4). However,
randomized controlled trials are needed for further
validation.
The limitations of this study are its relatively small
cohort size, short follow-up time, and data from a single study center. A prospective, multicenter, randomized clinical trial should be designed to further validate
the prognostic significance of CTCs in HCC.
To our knowledge, this is the first report to identify
the stem cell–like characteristics of EpCAMþ CTCs
and their prognostic significance using the standardized
CellSearch system in HCC patients. A preoperative
EpCAMþ CTC7.5 level of 2 is an independent prognostic indicator for recurrence in HCC patients undergoing curative resection. Monitoring dynamic changes
of perioperative CTCs may be a promising predictor
of the response of the therapeutic regimen. Eradicating
these cells might open a therapeutic avenue toward
preventing HCC recurrence.
References
1. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J
Clin 2010;60:277-300.
2. Cha C, Fong Y, Jarnagin WR, Blumgart LH, DeMatteo RP. Predictors
and patterns of recurrence after resection of hepatocellular carcinoma. J
Am Coll Surg 2003;197:753-758.
3. Pantel K, Brakenhoff RH, Brandt B. Detection, clinical relevance and
specific biological properties of disseminating tumour cells. Nat Rev
Cancer 2008;8:329-340.
4. Aktas B, Tewes M, Fehm T, Hauch S, Kimmig R, Kasimir-Bauer S.
Stem cell and epithelial-mesenchymal transition markers are frequently
overexpressed in circulating tumor cells of metastatic breast cancer
patients. Breast Cancer Res 2009;11:R46.
5. Raimondi C, Gradilone A, Naso G, Vincenzi B, Petracca A, Nicolazzo
C, et al. Epithelial-mesenchymal transition and stemness features in circulating tumor cells from breast cancer patients. Breast Cancer Res
Treat 2011;130:449-455.
6. Kim MY, Oskarsson T, Acharyya S, Nguyen DX, Zhang XH, Norton
L, et al. Tumor self-seeding by circulating cancer cells. Cell 2009;139:
1315-1326.
7. Clevers H. The cancer stem cell: premises, promises and challenges.
Nat Med 2011;17:313-319.
8. Allard WJ, Matera J, Miller MC, Repollet M, Connelly MC, Rao C,
et al. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases.
Clin Cancer Res 2004;10:6897-6904.
9. Yamashita T, Ji J, Budhu A, Forgues M, Yang W, Wang HY, et al.
EpCAM-positive hepatocellular carcinoma cells are tumor-initiating
1468
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
SUN, XU, YANG, ET AL.
cells with stem/progenitor cell features. Gastroenterology 2009;136:
1012-1024.
Yamashita T, Forgues M, Wang W, Kim JW, Ye Q, Jia H, et al.
EpCAM and alpha-fetoprotein expression defines novel prognostic subtypes of hepatocellular carcinoma. Cancer Res 2008;68:1451-1461.
Poon RT, Ng IO, Lau C, Yu WC, Yang ZF, Fan ST, et al. Tumor
microvessel density as a predictor of recurrence after resection of hepatocellular carcinoma: a prospective study. J Clin Oncol 2002;20:
1775-1785.
Bruix J, Sherman M. Management of hepatocellular carcinoma: an
update. HEPATOLOGY 2011;53:1020-1022.
Wittekind C. Pitfalls in the classification of liver tumors [in German].
Pathologe 2006;27:289-293.
Yang XY, Xu Y, Shi GM, Fan J, Zhou J, Ji Y, et al. Cytokeratin 10 and
cytokeratin 19: predictive markers for poor prognosis in hepatocellular
carcinoma patients after curative resection. Clin Cancer Res 2008;14:
3850-3859.
Llovet JM, Di Bisceglie AM, Bruix J, Kramer BS, Lencioni R, Zhu
AX, et al. Design and endpoints of clinical trials in hepatocellular carcinoma. J Natl Cancer Inst 2008;100:698-711.
Yang XR, Xu Y, Yu B, Zhou J, Qiu SJ, Shi GM, et al. High expression
levels of putative hepatic stem/progenitor cell biomarkers related to
tumour angiogenesis and poor prognosis of hepatocellular carcinoma.
Gut 2010;59:953-962.
Shah SA, Greig PD, Gallinger S, Cattral MS, Dixon E, Kim RD, et al.
Factors associated with early recurrence after resection for hepatocellular
carcinoma and outcomes. J Am Coll Surg 2006;202:275-283.
Reinholz MM, Kitzmann KA, Tenner K, Hillman D, Dueck AC, Hobday TJ, et al. Cytokeratin-19 and mammaglobin gene expression in circulating tumor cells from metastatic breast cancer patients enrolled in
North Central Cancer Treatment Group trials, N0234/336/436/437.
Clin Cancer Res 2011;17:7183-7193.
Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC,
et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 2004;351:781-791.
Kallergi G, Markomanolaki H, Giannoukaraki V, Papadaki MA, Strati
A, Lianidou ES, et al. Hypoxia-inducible factor-1alpha and vascular endothelial growth factor expression in circulating tumor cells of breast
cancer patients. Breast Cancer Res 2009;11:R84.
Camp RL, Dolled-Filhart M, Rimm DL. X-tile: a new bio-informatics
tool for biomarker assessment and outcome-based cut-point optimization. Clin Cancer Res 2004;10:7252-7259.
Mehes G, Witt A, Kubista E, Ambros PF. Circulating breast cancer
cells are frequently apoptotic. Am J Pathol 2001;159:17-20.
HEPATOLOGY, April 2013
23. Larson CJ, Moreno JG, Pienta KJ, Gross S, Repollet M, O’Hara SM,
et al. Apoptosis of circulating tumor cells in prostate cancer patients.
Cytometry A 2004;62:46-53.
24. Tung-Ping Poon R, Fan ST, Wong J. Risk factors, prevention, and
management of postoperative recurrence after resection of hepatocellular carcinoma. Ann Surg 2000;232:10-24.
25. Shah SA, Cleary SP, Wei AC, Yang I, Taylor BR, Hemming AW, et al.
Recurrence after liver resection for hepatocellular carcinoma: risk factors, treatment, and outcomes. Surgery 2007;141:330-339.
26. Sun YF, Yang XR, Zhou J, Qiu SJ, Fan J, Xu Y. Circulating tumor
cells: advances in detection methods, biological issues, and clinical relevance. J Cancer Res Clin Oncol 2011;137:1151-1173.
27. Kim H, Choi GH, Na DC, Ahn EY, Kim GI, Lee JE, et al. Human
hepatocellular carcinomas with ‘‘Stemness’’-related marker expression:
keratin 19 expression and a poor prognosis. HEPATOLOGY 2011;54:
1707-1717.
28. Takahashi-Yanaga F, Kahn M. Targeting Wnt signaling: can we safely
eradicate cancer stem cells? Clin Cancer Res 2010;16:3153-3162.
29. Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer 2009;9:265-273.
30. Chan SL, Mo FK, Johnson PJ, Hui EP, Ma BB, Ho WM, et al. New
utility of an old marker: serial-fetoprotein measurement in predicting
radiologic response and survival of patients with hepatocellular carcinoma undergoing systemic chemotherapy. J Clin Oncol 2009;27:
446-452.
31. Wang CC, Iyer SG, Low JK, Lin CY, Wang SH, Lu SN, et al. Perioperative factors affecting long-term outcomes of 473 consecutive patients
undergoing hepatectomy for hepatocellular carcinoma. Ann Surg Oncol
2009;16:1832-1842.
32. Cohen SJ, Punt CJ, Iannotti N, Saidman BH, Sabbath KD, Gabrail
NY, et al. Relationship of circulating tumor cells to tumor response,
progression-free survival, and overall survival in patients with metastatic
colorectal cancer. J Clin Oncol 2008;26:3213-3221.
33. Liu MC, Shields PG, Warren RD, Cohen P, Wilkinson M, Ottaviano
YL, et al. Circulating tumor cells: a useful predictor of treatment efficacy in metastatic breast cancer. J Clin Oncol 2009;27:5153-5159.
34. Ren ZG, Lin ZY, Xia JL, Ye SL, Ma ZC, Ye QH, et al. Postoperative
adjuvant arterial chemoembolization improves survival of hepatocellular
carcinoma patients with risk factors for residual tumor: a retrospective
control study. World J Gastroenterol 2004;10:2791-2794.
35. Yang XR, Xu Y, Yu B, Zhou J, Li JC, Qiu SJ, et al. CD24 is a novel
predictor for poor prognosis of hepatocellular carcinoma after surgery.
Clin Cancer Res 2009;15:5518-5527.