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Table S1. Primer pairs use for real-time quantitative RT-PCR Studies
Gene symbol
ABCG2
EpCAM
Nestin
CK7
CK14
CK19
CD44H
CD56
CD90
CD117
CD133
CD34
VEGF
PD-ECGF
beta-actin
Sequence 5’-3’
Amplicon size
F:5'CACCTTATTGGCCTCAGGAA3'
R:5'CCTGCTTGGAAGGCTCTATG3'
F:5'TTCTCAATGCAGGGTCTA3'
R:5'CCTTTATCTCAGCCTTCTC3'
F:5'AGCCAGATCGCTCAGGTCC3'
R:5'TGTTTGCAGCCGGGAGTT3'
F:5'CATCGAGATCGCCACCTACC3'
R:5'GCCACTGCTACTGCCACCA3'
F:5'GATGCCGAGGAATGGTTC3'
R:5'ATGCTGAGCTGGGACTGC3'
F:5'CGCCAAGATCCTGAGTGA3'
R:5'CAGTAACCTCGGACCTGCT3'
F:5'AAAGGAGCAGCACTTCAGGA3'
R:5'TGTGTCTTGGTCTCTGGTAGC3'
F:5'AACATCAGCAGCGAAGAA3'
R:5'AGGTACATGGACTGGGAGT3'
F:5'TGAAGGTCCTCTACTTATCCG3'
R:5'CACGAGGTGTTCTGAGCC3'
F:5'CAGCCCCTATCCTGGAAT3'
R:5'TTGCTTGAATGTTGGTCTTT3'
F:5'CCGCAGGAGTGAATCTTT3'
R:5'AGGACTCGTTGCTGGTGA3'
F:5'GTCTACTGCTGGTCTTGGC3'
R:5'AGTGCAATCAGGGTCTTTT3'
F:5’CGCAAGAAATCCCGGTATAAGT3’
R:5’TGCTTTCTCCGCTCTGAGC3’
F:5'GGCTGCTGTATCGTGGGT3'
R:5'TGAGAATGGAGGCTGTGATG3'
F:5'TTGTTACAGGAAGTCCCTTGCC3'
R:5'ATGCTATCACCTCCCCTGTGTG3'
206bp
1
136bp
120bp
118bp
152bp
158bp
128bp
161bp
169bp
157bp
133bp
157bp
116bp
118bp
101bp
Table S2. Antibodies and TMA scoring criteria
Antibody
CK7
1
Manufacturer
DAKO,
Denmark
CK19 1
DAKO,
Denmark
OV6 2
R&D , USA
Nestin 3
Santa Cruz，
USA
CD133 4, 5
Abgent, USA
CD44H 6
Bender
medsystems
ABCG2 7
CHEMIC,
USA
EPCAM 8
ABR
CD34 9, 10
DAKO,
Denmark
VEGF11
Calbiochem,
U.S.A
PD-EGGF 12
Santa Cruz,
USA
Staining
pattern
Cytoplasmic
and
membrane
Cytoplasmic
and
membrane
Cytoplasmic
And
membrane
Cytoplasmic
and
membrane
Cytoplasmic
and
membrane
Cytoplasmic
and
membrane
Cytoplasmic
and
membrane
Cytoplasmic
and
membrane
cytoplasmic
Cytoplasmic
and
membrane
Cytoplasmic
and
membrane
Dilution
Time§
1:100
Score†
Species
negative
positive
1h
<5%
≥5%
Mouse
monoclonal
1:100
1h
<5%
≥5%
Mouse
monoclonal
1:100
Overnight
<5%
≥5%
Mouse
monoclonal
1:100
Overnight
<5%
≥5%
Mouse
monoclonal
1:50
Overnight
≤1%
>1%
Mouse
monoclonal
1:100
Overnight
≤10%
>10%
Mouse
monoclonal
1:40
Overnight
≤10%
>10%
Mouse
monoclonal
1:40
Overnight
<5%
≥5%
Mouse
monoclonal
1:100
Overnight
≤55.5
>55.5 ‡
Mouse
monoclonal
1:100
Overnight
<30%
≥30%
Rabbit
monoclonal
1:100
Overnight
<5%
≥5%
Mouse
monoclonal
Abbreviations: CK19, Cytokeratin 19; CK7, Cytokeratin 7; MVD, microvessel density; VEGF, vascular
endothelial growth factor; PD-ECGF, platelet derived endothelial cell growth factor.
§ 1h was incubated at room temperature and overnight was at 4 ℃
† The staining intensity of positive tumor cell was moderate or strong.
‡ Any brownstained endothelial cell or endothelial cell cluster that was clearly separated from adjacent
microvessels, tumor cells, and connective elements was counted as one microvessel, irrespective of the
presence of a vessel lumen. MVD of each cylinder (0.785mm2/field) was examined. The cut-off value
was the median MVD of whole population.
2
Reference
1
Durnez A, Verslype C, Nevens F, et al. The clinicopathological and prognostic relevance of cytokeratin 7 and 19
expression in hepatocellular carcinoma. A possible progenitor cell origin. Histopathology 2006;49:138-51.
2
Libbrecht L, Desmet V, Van Damme B, et al. The immunohistochemical phenotype of dysplastic foci in human
liver: correlation with putative progenitor cells. J Hepatol 2000;33:76-84.
3
Ikota H, Kinjo S, Yokoo H, et al. Systematic immunohistochemical profiling of 378 brain tumors with 37
antibodies using tissue microarray technology. Acta Neuropathol 2006;111:475-82.
4
Ma S, Chan KW, Hu L, et al. Identification and characterization of tumorigenic liver cancer stem/progenitor cells.
Gastroenterology 2007;132:2542-56.
5
Zeppernick F, Ahmadi R, Campos B, et al. Stem cell marker CD133 affects clinical outcome in glioma patients.
Clin Cancer Res 2008;14:123-9.
6
Yang GH, Fan J, Xu Y, et al. Osteopontin combined with CD44, a novel prognostic biomarker for patients with
hepatocellular carcinoma undergoing curative resection. Oncologist 2008;13:1155-65.
7
Yoh K, Ishii G, Yokose T, et al. Breast cancer resistance protein impacts clinical outcome in platinum-based
chemotherapy for advanced non-small cell lung cancer. Clin Cancer Res 2004;10:1691-7.
8
Yamashita T, Forgues M, Wang W, et al. EpCAM and alpha-fetoprotein expression defines novel prognostic
subtypes of hepatocellular carcinoma. Cancer Res 2008;68:1451-61.
9
Sun HC, Tang ZY, Li XM, et al. Microvessel density of hepatocellular carcinoma: its relationship with prognosis. J
Cancer Res Clin Oncol 1999;125:419-26.
10
Poon RT, Ng IO, Lau C, et al. Tumor microvessel density as a predictor of recurrence after resection of
hepatocellular carcinoma: a prospective study. J Clin Oncol 2002;20:1775-85.
11
Lee TK, Poon RT, Yuen AP, et al. Regulation of angiogenesis by Id-1 through hypoxia-inducible
factor-1alpha-mediated vascular endothelial growth factor up-regulation in hepatocellular carcinoma. Clin Cancer Res
2006;12:6910-9.
12
Zhou J, Tang ZY, Fan J, et al. Capecitabine inhibits postoperative recurrence and metastasis after liver cancer
resection in nude mice with relation to the expression of platelet-derived endothelial cell growth factor. Clin Cancer Res
2003;9:6030-7.
3
Table S3. Descriptive statistics of immunohistochemical variables in cohort 2
(n=314)
Immunohistochemical variables
CK19
CK7
ABCG2
CD133
Nestin
CD44
OV6
EPCAM
MVD
VEGF
PD-ECGF
HSCs/HPCs profile
predictive model
The simplified model
Number
Percentage (%)
Negative
Positive
Negative
Positive
Negative
Positive
Negative
Positive
Negative
Positive
Negative
Positive
Negative
Positive
Negative
Positive
≤55.5
＞55.5
Negative
Positive
274
40
251
63
247
67
233
81
226
88
223
91
174
140
264
50
157
157
118
196
87.26
12.74
79.94
20.06
78.66
21.34
74.20
25.80
71.97
28.03
71.02
28.98
55.41
44.59
84.08
15.92
50.00
50.00
37.60
62.40
Negative
112
35.67
Positive
Low
High
Low risk
Moderate risk
High risk
202
163
151
75
138
101
64.33
51.91
48.09
23.89
43.95
32.16
Abbreviations: HSCs, hepatic stem cells; HPCs, hepatic progenitor cells; CK19,
cytokeratin 19; CK7, cytokeratin 7; MVD, microvessel density; VEGF, vascular endothelial
growth factor; PD-ECGF, platelet derived endothelial cell growth factor.
4
Table S4. Multivariate analyses of HSCs/HPCs
clinicopathological characteristics in cohort 2(n=314)
profile
OS
predictive
model
with
RFS
Variables
Hazard ratio
(95% CI)
P value
Hazard ratio
(95% CI)
P value
GGT (>54 U/l vs.≤54 U/l)
Tumor encapsulation
(none vs. complete)
Tumor differentiation (III-IV vs. I-II)
Tumor size (>5 cm vs.≤5 cm)
Tumor number (multiple vs.single)
Vascular invasion (yes vs.no)
HSCs/HPCs predictive model
(HSCs/HPCshigh vs. HSCs/HPCslow)
1.96(1.45-2.66)
<0.0001
1.68(1.23-2.30)
0.001
1.18(0.88-1.59)
0.263
n.a.
1.28(0.94-1.75)
1.42(1.06-1.92)
1.58(1.09-2.27)
1.66(1.14-2.40)
0.119
0.020
0.014
0.008
n.a.
n.a.
1.52(1.02-2.27)
1.61(1.07-2.43)
0.038
0.023
2.09(1.55-2.82)
<0.0001
2.39(1.75-3.27)
<0.0001
Abbreviations: HSCs, hepatic stem cells; HPCs, hepatic progenitor cells; AFP,
alpha-fetoprotein; GGT, serum gammaglutamyl transferase; RFS, Relapse-free survival; OS,
overall survival; n.a., not applicable.
Multivariate analysis, Cox proportional hazards regression model. Variables were adopted for
their prognostic significance by univariate analysis.
5
Table
S5.
Correlation
between
HSCs/HPCs
clinicopathological characteristics in cohort 2(n=314)
Patients
Age(y) †
HBsAg
HCV‡
Liver cirrhosis
Child-Pugh score ‡
ALT(U/l) †
GGT(U/l) †
AFP(ng/ml) †
Tumor encapsulation§
Tumor size (cm)
Tumor number
Vascular invasion
TNM stage
Tumor differentiation
CLIP stage
Prophylactic TACE
VEGF
PD-ECGF
MVD†
Recurrence time(m)
Recurrence types
model
and
HSCs/HPCs predictive model
Variables
Sex
predictive
Female
Male
Mean±SD
Negative
Positive
Negative
Positive
No
Yes
A
B
Mean±SD
Mean±SD
Mean±SD
complete
no
≤5
>5
Single
Multiple
No
Yes
I
II-III
I-II
III-IV
0+1
2+3+4
No
Yes
negative
Positive
negative
Positive
Mean±SD
≤24
>24
Type I
low
high
163
26
137
52.7±10.3
30
133
161
2
15
148
163
0
46.8±53.7
84.0±71.9
625.8±1183.5
95
66
85
78
141
22
137
26
119
44
120
43
128
35
95
68
72
91
63
100
78.0±89.2
34
35
46
151
22
129
52.0±11.6
25
126
148
3
11
140
149
2
46.9±58.6
75.2±80.4
512.47±1103.9
79
71
86
65
126
25
130
21
109
42
107
44
120
31
97
54
46
105
49
102
110.3±118.4
61
39
72
6
P value
0.709
0.616
0.667
0.674
0.538
0.230
0.987
0.304
0.382
0.26
0.393
0.448
0.612
0.871
0.585
0.838
0.279
0.012
0.252
0.007
0.131
0.660
Type II
9
13
Type III
14
15
Abbreviations: HSCs, hepatic stem cells; HPCs, hepatic progenitor cells; AFP,
alpha-fetoprotein; ALT, alanine aminotransferase; GGT, gammaglutamyl transferase;
TACE, transcatheter arterial chemoembolization; MVD, microvessel density; TNM,
tumor-node-metastasis; CLIP, Cancer of Liver Italian Program.
†Student’s t-test was used for comparison between groups. ‡Fisher’s exact tests;
Chi-square tests for all the other analyses.
§ 3 cases without capsulation information were not calculated.
7
Table S6. The ROC analyses of Variables for Recurrence and Death
Recurrence
Variables
Death
AUC
95%CI
AUC
95%CI
Tumor size
0.507
0.451 to 0.562
0.550
0.494 to 0.606
Tumor number
0.53
0.491 to 0.569
0.564
0.527 to 0.600
Vascular invasion
0.517
0.478 to 0.557
0.550
0.513 to 0.588
Edmondson stage
0.527
0.477 to 0.576
0.562
0.514 to 0.611
Tumor encapsulation
0.474
0.419 to 0.530
0.542
0.485 to 0.598
TNM stage
0.556
0.507 to 0.605
0.600
0.553 to 0.646
Clip stage
0.516
0.471 to 0.561
0.547
0.503 to 0.591
GGT
0.55
0.495 to 0.605
0.620
0.565 to 0.675
AFP
0.492
0.436 to 0.547
0.511
0.454 to 0.568
Nestin
0.575
0.526 to 0.623
0.552
0.502 to 0.601
CD133
0.554
0.506 to 0.602
0.587
0.540 to 0.633
CD44
0.622
0.574 to 0.669
0.600
0.552 to 0.647
HPCs/HSCs profile
0.62
0.566 to 0.674
0.660
0.607 to 0.713
MVD
0.689
0.638 to 0.740
0.624
0.570 to 0.679
The simplified model
0.751
0.702 to 0.800
0.710
0.657 to 0.763
Abbreviations: ROC, receiver operating characteristic; AUC, area under the curve; 95% CI; 95%
confidence interval; AFP, alpha-fetoprotein; GGT, gammaglutamyl transferase; MVD, microvessel
density; TNM, tumor-node-metastasis; CLIP, Cancer of Liver Italian Program; HSCs, hepatic stem cells;
HPCs, hepatic progenitor cells.
8
Table S7. Univariate analyses of factors associated with survival and recurrence in
cohort 3 (n=73)
OS
Variables
Sex (male vs. female)
Age (>52y vs.≤52y)
HBsAg (positive vs.negative)
HCV (positive vs.negative)
Child–Pugh score (B vs.A)
Liver cirrhosis (yes vs.no)
GGT (>54 U/l vs.≤54 U/l)
ALT (>75 U/l vs.≤75 U/l)
AFP (>20 ng/ml vs.≤20 ng/ml)
Tumor differentiation (III-IV vs.I-II)
Tumor
encapsulation
vs.complete)
(none
Tumor size (>5 cm vs.≤5 cm)
Tumor number (multiple vs.single)
Vascular invasion (yes vs.no)
TNM stage (II+III vs.I)
CLIP stage 2+3+4 vs 0+1
Prophylactic TACE(yes vs.no)
CD44 (positive vs.negative)
Nestin (positive vs.negative)
RFS
Hazard ratio
P
Hazard ratio
(95% CI)
value
(95% CI)
1.45(0.19-10.8
6)
0.50(0.20-1.22
)
0.94(0.31-2.80
)
0.05(0-325175
.8)
8.73(1.94-39.3
2)
0.86(0.29-2.59
)
4.61(1.54-13.8
1)
1.18(0.34-4.02
)
4.67(1.37-15.9
6)
1.31(0.52-3.28
)
1.54(0.64-3.72
)
4.96(1.80-13.6
8)
0.91(0.31-2.74
)
3.30(1.20-9.09
)
3.09(1.03-9.25
)
7.03(2.78-17.8
1)
0.82(0.34-1.97
)
2.82(1.15-6.92
)
2.06(0.82-5.15
0.71
5
0.12
8
0.90
8
0.70
6
0.00
5
0.79
5
0.00
6
0.79
5
0.01
4
0.56
8
0.33
7
0.00
2
0.87
3
0.02
1
0.04
4
0.00
0
9
0.66
0.02
3
0.12
1.70(0.23-12.67)
0.63(0.27-1.45)
1.01(0.34-3.00)
0.05(0.00-81724.
00)
4.41(0.57-34.11)
0.42(0.17-1.03)
1.66(0.71-3.90)
0.64(0.15-2.74)
2.95(1.09-8.03)
1.12(0.46-2.75)
0.85(0.37-2.00)
2.85(1.19-6.81)
0.56(0.17-1.91)
2.48(1.01-6.10)
2.85(1.05-7.73)
2.61(1.11-6.14)
1.29(0.55-3.01)
2.47(1.03-5.91)
2.28(0.96-5.45)
P
valu
e
0.60
3
0.27
5
0.98
1
0.67
9
0.15
5
0.05
8
0.24
1
0.54
6
0.03
4
0.80
3
0.71
7
0.01
8
0.35
6
0.04
8
0.04
0
0.02
8
0.56
1
0.04
2
0.06
CD133 (positive vs.negative)
MVD (>55.5 vs.≤55.5)
)
0.63(0.23-1.73
)
2.68(0.97-7.38
)
The simplified model
moderate risk vs. low risk
high risk vs. low risk
2.02(0.41-10.0
3)
4.57(1.02-20.4
5)
5
0.37
0
0.05
6
0.06
6
0.38
8
0.04
7
2.14(0.93-4.94)
3.40(1.25-9.24)
1.76(0.34-9.09)
7.01(1.60-30.80)
3
0.07
5
0.01
7
0.00
3
0.49
8
0.01
0
Abbreviations: ALT, alanine aminotransferase; GGT, serum gammaglutamyl transferase;
AFP, alpha-fetoprotein; CK19, cytokeratin 19; CK7, cytokeratin 7; MVD, microvessel
density; VEGF, vascular endothelial growth factor; PD-ECGF, platelet derived endothelial
cell growth factor; TNM, tumor-node-metastasis; CLIP, Cancer of Liver Italian Program;
95%CI, 95% confidence interval; RFS, Relapse-free survival; OS, overall survival.
Univariate analysis, Cox proportional hazards regression model.
10