Download of Tumor Origin Influence in Colorectal and Renal Cell Carcinoma

Published OnlineFirst June 19, 2013; DOI: 10.1158/1078-0432.CCR-12-3847
Characteristics and Clinical Impacts of the Immune Environments
in Colorectal and Renal Cell Carcinoma Lung Metastases: Influence
of Tumor Origin
Romain Remark, Marco Alifano, Isabelle Cremer, et al.
Clin Cancer Res 2013;19:4079-4091. Published OnlineFirst June 19, 2013.
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Published OnlineFirst June 19, 2013; DOI: 10.1158/1078-0432.CCR-12-3847
Clinical
Cancer
Research
Human Cancer Biology
Characteristics and Clinical Impacts of the Immune
Environments in Colorectal and Renal Cell Carcinoma Lung
Metastases: Influence of Tumor Origin
Romain Remark1,2,3,4, Marco Alifano3,5, Isabelle Cremer1,2,3, Audrey Lupo1,2,3,4,5,
Marie-Caroline Dieu-Nosjean1,2,3, Marc Riquet3,6, Lucile Crozet1,2,3, Hanane Ouakrim1,2,3, Jeremy Goc1,2,3,
jou2,8, Laure Gibault3,9, Virginie Verkarre3,10, Jean-François Re
gnard3,5,
lie Cazes3,6, Jean-François Fle
Aure
5
3,6
1,2,3
1,2,3
, Catherine Sautes-Fridman
,
Olivier-Nicolas Pages , Stephane Oudard , Bernhard Mlecnik
Wolf-Herman Fridman1,2,3,7, and Diane Damotte1,2,3,5
Abstract
Purpose: If immune cells are involved in tumor surveillance and have a prognostic impact in most
primary tumors, little is known about their significance in metastases. Because patients’ survival is
heterogeneous, even at metastatic stages, we hypothesized that immune cells may be involved in the
control of metastases. We therefore characterized the tumor immune microenvironment and its
prognostic value in colorectal and renal cell carcinoma (RCC) metastases, and compared it to primary
tumors.
Experimental Design: We analyzed by immunohistochemistry (n ¼ 192) and qPCR (n ¼ 32) the
immune environments of colorectal carcinoma and RCC lung metastases.
Results: Metastases from colorectal carcinoma and RCC have different immune infiltrates. Higher
densities of DC-LAMPþ mature dendritic cells (P < 0.0001) and lower densities of NKp46þ NK cells (P <
0.0001) were observed in colorectal carcinoma as compared to RCC metastases, whereas densities of T cells
were similar. High densities of CD8þ and DC-LAMPþ cells correlated with longer overall survival (OS) in
colorectal carcinoma (P ¼ 0.008) and shorter OS in RCC (P < 0.0001). High NK-cell densities were
associated with improved survival in RCC (P ¼ 0.002) but not in colorectal carcinoma. Densities of immune
cells correlated significantly from primary to relapsing metastases for the same patient. A TH1 orientation
was found in colorectal carcinoma metastases, whereas a heterogeneous immune gene expression was found
in RCC metastases.
Conclusions: Our results show a major prognostic value of the immune pattern (CD8þ/DC-LAMPþ cell
densities) in colorectal carcinoma and RCC, reproducible from primary to metastatic tumors, although with
opposite clinical impacts, and highlight the role of the tumor cell in shaping its immune environment. Clin
Cancer Res; 19(15); 4079–91. 2013 AACR.
et de la Recherche
Authors' Affiliations: 1Institut National de la Sante
dicale (INSERM), U872, Centre de Recherche des Cordeliers; 2UniMe
3
Paris Des Pierre et Marie Curie-Paris 6, UMRS 872; Universite
versite
Denis Diderot-Paris 7; 5Services
cartes-Paris 5, UMRS 872; 4Universite
^ pital Ho
^ tel Dieu;
d'anatomie-pathologique et de chirurgie thoracique, Ho
6
Services d'anatomie-pathologique, oncologie et de chirurgie thoracique;
7
^ pital Europe
en Georges Pompidou;
Service d'Immunologie Biologique, Ho
8
^ pital Saint-Antoine; 9Service d'anaService d'anatomie-pathologique, Ho
10
^ pital Cochin; and Service d'anatomie-patholotomie-pathologique, Ho
^ pital Necker-Enfants Malades, AP-HP, Paris, France
gique, Ho
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
W.-H. Fridman and D. Damotte contributed equally to this article.
Corresponding Author: Diane Damotte, INSERM U872, Centre de
decine, 75006 Paris,
Recherche des Cordeliers, 15 rue de l'Ecole de Me
France. Phone: 33-1-42-34-87-12; Fax: 33-1-42-34-86-41; E-mail:
[email protected]
doi: 10.1158/1078-0432.CCR-12-3847
2013 American Association for Cancer Research.
Introduction
Immune cells are found in human solid tumors, and the
immune pattern of the tumor microenvironment is a major
predictor of patient survival in a large array of primary
tumors (1). Thus, a high density of T cells with a TH1 and
CD8þ T cells cytotoxic orientation or of mature dendritic
cells (DC) are beneficial in most cancers, especially in
colorectal (2–4), lung (5), breast (6), gastric (7), pancreatic
(8), urothelial (9), hepatocellular (10), esophageal (11),
ovarian cancer (12) and melanoma (13), with the exception
of renal cell carcinoma (RCC) in which high densities of
CD8þ and CD45ROþ cells are associated with poor prognosis (14, 15).
Even if metastatic spreading is the main cause of death
by cancer (16), metastatic patients have heterogeneous
survival (17). A classical view of cancer progression is that
genetic modifications (18) may allow malignant cells to
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Published OnlineFirst June 19, 2013; DOI: 10.1158/1078-0432.CCR-12-3847
Remark et al.
Translational Relevance
This article demonstrates for the first time, in a large
cohort of patients with lung metastasis from 2 different
primary tumors, colorectal and renal cell carcinoma,
that densities of CD8þ T cells and DC-LAMPþ mature
dendritic cells ("immune pattern"), evaluated in paraffin sections, were independent prognostic factors of
patients’ survival, and stronger prognosticators than
currently evaluated clinical and pathological parameters. Furthermore, tumor immune environment is
reproduced throughout cancer disease, from primary
tumor to relapsing metastasis. This finding is the first
important step for further extensive studies on the role
of the tumor cells in shaping their own immune environment and the patients’ outcome.
A
escape local and systemic immune control (19) and
consequently invade and metastasize in distant organs.
This hypothesis would predict that the immune microenvironment in metastatic sites should be poor and have
no impact on clinical outcome. Only a limited number of
studies have reported the presence of immune cells in
metastatic lesions. They showed that high densities of
CD8þ T cells were associated with longer survival in
colorectal carcinoma (20) and ovarian cancer (21), and
potential response to chemotherapy in liver metastases
from colorectal carcinoma (22). Another question, which
remains largely unanswered, concerns the respective roles
of the malignant cells and the seeding organ in shaping
the immune microenvironment.
We therefore analyzed the immune environment of colorectal carcinoma and RCC metastases seeded in a same
organ, the lung, compared coincident and relapsing
C
CD8+ T cells:
CD8+ and DC-LAMP+ cells:
P = 0.039
1
0.8
Overall survival
Overall survival
0.8
0.6
0.4
0.2
B
CD8 hi / DC-LAMP hi (n = 67)
CD8 lo / DC-LAMP hi
or CD8 hi / DC-LAMP lo (n = 53)
CD8 lo / DC-LAMP lo (n = 20)
0
20
40
60
80
100
Hi
71
53
40
24
12
4
Lo
69
52
30
15
5
2
Time (months) 0
120
Hi/Hi
At risk
patients
D
DC-LAMP+ mature DC:
67
20
40
60
80
100
51
39
24
13
4
MIX
53
42
24
9
3
1
Lo/Lo
20
12
7
6
2
1
120
NKp46+ NK cells:
P = 0.001
1
P = 0.12
1
0.8
0.8
Overall survival
Overall survival
0.4
CD8 lo (n = 69)
Time (months) 0
0.6
0.4
0.2
DC-LAMP
Time (months) 0
lo
0.6
0.4
0.2
DC-LAMP hi (n = 116)
0
At risk
patients
0.6
0.2
CD8 hi (n = 71)
0
At risk
patients
P = 0.008
1
NKp46 hi (n = 58)
(n = 24)
NKp46 lo (n = 26)
0
20
40
60
80
100
Hi
116
91
62
33
16
5
Lo
24
14
8
6
2
1
120
Time (months) 0
At risk
patients
20
40
60
80
100
Hi
58
49
39
23
14
5
Lo
26
17
8
3
0
0
120
þ
þ
þ
Figure 1. Prognostic value of the densities of CD8 T cells, DC-LAMP mature dendritic cells, and NKp46 NK cells in lung metastases from
colorectal carcinoma. Kaplan–Meier curves for the duration of OS according to a separated (A, B) and combined (C) analysis of CD8þ and DC-LAMPþ densities
in colorectal carcinoma lung metastases. D, Kaplan–Meier curves for the duration of OS according to the densities of NKp46þ cells in colorectal
carcinoma lung metastases (n ¼ 84). The numbers of at risk patients according to a separated and combined analysis of CD8þ and DC-LAMPþ densities and
NKp46þ cells densities were given. Statistical comparison was conducted by the log-rank test and all OS log-rank P values were corrected using the
formula proposed by Altman and colleagues.
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Clin Cancer Res; 19(15) August 1, 2013
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Published OnlineFirst June 19, 2013; DOI: 10.1158/1078-0432.CCR-12-3847
In Situ Immune Reaction in Lung Metastases
E
High
expression
Low expression
–2.0
1:1
2.0
Mean(ΔCt) P = 0.014
CD3E
CD4
CD8A
CD68
Immune cell
populations
Mean(ΔCt) P = 0.04
IFNG
IL12A
IL12B
1L18
TBX21
LTA
TH1
orientation
Mean(ΔCt) P = 0.42
Figure 1. (Continued ) E, expression
of genes related to immune cell
populations, TH1/TH2 orientations,
inflammation, angiogenesis,
immuno-suppression, cytotoxicity,
chemokines/chemokine receptors
according to the densities of CD8þ
and DC-LAMPþ cells (high/high vs.
low/low) in lung metastases from
colorectal carcinoma. Expression
levels of genes were determined
using threshold cycle (Ct) values
normalized to actin B [ACTB] (DCt).
We used Mann–Whitney test to
identify genes with significantly
different levels of expression
among patient groups (high vs. low
CD8þ/DC-LAMPþ densities).
, P < 0.05 for individual gene
expression.
IL4
IL5
IL10
IL13
TH2
orientation
Mean(ΔCt) P = 0.39
C3
FN1
IL3
IL6
IL7
TNF
CSF1
IL1A
IL8
IL1B
IL17
PTGS2
SELE
CSF3
STAT3
Inflammation
*
mean(ΔCt) P = 0.13
ACE
CD34
VEGF
Angiogenesis
mean(ΔCt) P = 0.23
Immunosuppression
TGFB1
CTLA4
IL10
*
*
mean(ΔCt) P = 0.032
GLNY
GZMB
PRF1
IL15
Cytotoxicity
mean(ΔCt) P = 0.03
CCL2
CCL3
CCL5
CCR2
CCR5
Chemokines/
chemokine
receptors
CCL19
CCR4
CCR7
CXCL10
CXCL11
CXCR3
High CD8+/DC-LAMP+
densities
metastases in the lung, and the primary tumor from the
same patients, and determined their clinical impacts.
We report that tumor cells induce a characteristic and
reproducible immune pattern in the primary and metastatic
tumors, supporting the hypothesis that the malignant cells,
rather than the host organ, shape their microenvironment.
We found that a high infiltration by DC-LAMPþ mature
dendritic cells and CD8þ T cells is a major predictor of good
survival in lung metastases from colorectal carcinoma,
whereas it is associated with poor survival in lung metastases
from RCC. This shows that the immune microenvironment
www.aacrjournals.org
Low CD8+/DC-LAMP+
densities
pattern remains a major prognostic factor even in advanced
cancer stages, but with different consequences depending
on the origin of the primary tumor. Altogether our results
suggest a strong influence of the tumor origin on the
immune environment characteristics and clinical impact.
Patients and Methods
Patients
We constituted a retrospective and unselected cohort of
140 patients with colorectal carcinoma lung metastasis
operated at H^
otel-Dieu hospital between 2000 and 2010
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Remark et al.
Table 1. Univariate and multivariate Cox proportional hazards analyses for OS according to clinical
parameters and immune cell densities in colorectal carcinoma and RCC lung metastases
Univariate analyses
Colorectal carcinoma
lung metastases
RCC lung metastases
Multivariate analyses
Variable
HR
95% CI
P value
HR
Stage (stage 3 þ 4 vs. stage 1 þ 2)
Presence of extrathoracic metastases
(yes vs. no)
Completeness of resection (R1 vs. R0)
CEA level (5 ng/mL vs. <5 ng/mL)
NK cells (high vs. low)
1.68
1.56
(0.88–3.20)
(0.88–2.75)
0.116
0.128
Not included in the
multivariate analysis
2.49
1.55
0.58
(0.77–8.05)
(0.86–2.82)
(0.28–1.16)
0.129
0.148
0.123
Thoracic lymph node invasion (yes vs. no)
Number of metastases (>2 vs. 2)
Immune pattern (high/mix/low)
1.49
1.84
0.54
(0.60–3.66)
(1.03–3.28)
(0.39–0.76)
0.0503
0.0405
0.0002
1.80
2.17
0.54
Initial Fuhrman nuclear grade (3 þ 4 vs.
1 þ 2)
Time from lung metastasis diagnosis to
surgery (>1 year vs. 1year)
Number of metastases (multiple vs. 1)
Presence of extrathoracic metastases
(yes vs. no)
Completeness of resection (R1 vs. R0)
Alkaline phosphatase (>80 U/L vs. 80 U/L)
Neutrophils (>7,500/mm3 vs. 7,500/mm3)
Platelets (>400,000/mm3 vs. 400,000/mm3)
1.32
(0.56–3.11)
0.533
Not included in the
multivariate analysis
1.77
(0.83–3.76)
0.142
1.30
1.34
(0.61–2.79)
(0.51–3.55)
0.501
0.555
1.40
1.52
0.87
0.80
(0.42–4.65)
(0.75–4.33)
(0.29–2.62)
(0.23–2.82)
0.587
0.682
0.805
0.728
0.35
2.23
(0.16–0.74)
(1.02–4.86)
0.0064
0.0435
0.33
1.74
(0.14–0.73)
(0.47–6.46)
0.0067
0.407
1.92
2.26
(0.91–4.05)
(0.96–5.33)
0.086
0.061
2.41
1.00
(0.81–7.17)
(0.34–2.67)
0.113
0.935
0.46
2.68
(0.22–0.95)
(1.58–4.57)
0.037
0.00028
0.32
2.70
(0.14–1.03)
(1.37–5.29)
0.0579
0.0039
a
DFI (1 year vs. <1 year)
Metastases at presentation (synchronous
vs. metachronous)
Thoracic lymph node invasion (yes vs. no)
Hemoglobin (men: <13 g/dL vs. 13 g/dL
and women: <12 g/dL vs. 12 g/dL)
NK cells (high vs. low)
Immune pattern (high/mix/low)
95% CI
(0.87–3.72)
(1.20–3.93)
(0.39–0.75)
P value
0.115
0.011
0.0003
NOTE: To be able to conduct regression with a categorical variable, they were coded before entered into the Cox model. Parameters
with significant impact on survival appear in bold.
a
The stage was determined by pathologic examination at the time of diagnosis. None of the variables violated the proportional hazards
assumption.
and 52 patients with RCC lung metastasis, operated at
H^
otel-Dieu or Laennec/H^
opital Europeen Georges Pompidou hospitals between 1992 and 2010. In the RCC series,
51 of 52 patients were treated with radical nephrectomy and
1 with partial nephrectomy. None of the patients had signs
of local recurrence of primary tumor. We also analyzed
25 colorectal carcinoma and 24 RCC primary tumors from
the same patients, operated at Saint-Antoine, Cochin, or
Necker-Enfants Malades hospitals between 1987 and 2008.
In addition, 14 coincident and 12 recurrent colorectal carcinoma lung metastases from the same patients were studied. Altogether, 218 lung metastases from 192 patients were
analyzed.
Among these 192 patients, 32 frozen samples of lung
metastases were available for patients with colorectal carcinoma (n ¼ 19) or RCC (n ¼ 13).
4082
Clin Cancer Res; 19(15) August 1, 2013
Baseline characteristics of these patients are summarized
in Supplementary Tables S1 and S2.
All experiments were conducted with the agreement of
the Ile de France II ethics committee (no. 2012-0612).
Immunohistochemistry
For each tumor, 2 observers (R. Remark and D. Damotte,
A. Lupo, A. Cazes, L. Gibault, or V. Verkarre, expert pathologists) selected the tumor section containing the highest
density of immune cells on hematoxylin and eosin stained
slides. Serial 5-mm formalin-fixed and paraffin-embedded
tissue sections were stained with autostainer Link 48 (Dako).
Tissue sections were incubated with primary antibodies
[CD3 polyclonal antibody (Dako), CD8 (SP16, Springbioscience), CD20 (L26, Dako), DC-LAMP (1010.01, Dendritics), granzyme B (11F1, Novocastra), NKp46 (195314,
Clinical Cancer Research
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Published OnlineFirst June 19, 2013; DOI: 10.1158/1078-0432.CCR-12-3847
In Situ Immune Reaction in Lung Metastases
A
Coincident metastases:
Relapsing metastases:
t = 1–9
months
C
R = 0.643
ns
Number of DC-LAMP+ cells/mm2
3,000
2,000
1,000
0
rs
Fi
E
d
on
sid
tm
0
s
ap
a
et
is
re
Lung
metastasis
R = 0.895
ns
rs
Fi
d
on
sid
tm
F
a
et
is
re
100
G
*
R = 0.659
ns
15
8,000
6,000
4,000
2,000
0
10
5
C
d
on
sid
c
Se
M
-L
C
CR
tm
ps
la
a
et
is
re
as
st
rs
Fi
e
is
as
st
a
et
M
H
R = 0.656
ns
800
600
400
200
0
0
T
-P
CR
e
e
id
ts
M
R = 0.693
10,000
200
rs
Fi
as
st
rs
Fi
s
ap
l
s
a
et
c
Se
s
ta
300
0
e
is
e
e
id
ts
M
Primary tumor versus metastases:
Primary
tumor
5
as
st
rs
Fi
10
l
s
a
et
c
Se
s
ta
R = 0.696
ns
400
15
e
is
e
e
id
ts
D
R = 0.580
ns
Number of DC-LAMP+ cells/mm2
Number of CD8+ cells/mm2
4,000
R = 0.614
ns
Metastasis
relapse
Number of NKp46+ cells/mm2
R = 0.644
ns
Number of CD8+ cells/mm2
B
First
metastasis
Second
side
Number of NKp46+ cells/mm2
First
side
t = 14–52
months
T
-P
C
CR
M
-L
C
CR
PT
C-
CR
LM
C-
CR
þ
þ
þ
Figure 2. CD8 T cells, DC-LAMP mature dendritic cells, and NKp46 NK cell densities in coincident or relapsing metastases and in primary
colorectal cancer. A, surgical treatment for coincident and relapsing colorectal carcinoma lung metastases. B–D, coincident or relapsing metastases have the
same densities of CD8þ, DC-LAMPþ, and NKp46þ cells. E, surgical treatment for primary colorectal carcinoma and their lung metastases. F–H,
colorectal carcinoma primary tumors were more infiltrated by CD8þ cells than lung metastases, but have similar densities of DC-LAMPþ and NKp46þ cells.
R values show the positive correlations (0.5 < R < 0.9 and P < 0.05, Spearman test) between coincident metastases, relapsing metastases, primary tumors,
and associated metastases according to the CD8þ, DC-LAMPþ, and NKp46þ cell densities. PT, primary tumor; LM, lung metastasis; ns, not significant;
, P < 0.05 (Wilcoxon matched pairs test).
R&D Systems), or PNAd (MECA-79, BD Pharmingen)]
followed by secondary antibodies coupled with biotin
or alkaline phosphatase. Biotinylated antibodies were coupled with streptavidin-peroxidase and peroxidase activity
www.aacrjournals.org
was revealed using 3-amino-9-ethylcarbazole substrate
(Vector Laboratories). Alkaline phosphatase activity was
revealed using alkaline phosphatase substrate III (Vector
Laboratories).
Clin Cancer Res; 19(15) August 1, 2013
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Remark et al.
A
C
CD8+ T cells:
CD8+ and DC-LAMP+ cells:
P = 0.03
1
0.8
Overall survival
0.8
Overall survival
P < 0.0001
1
0.6
0.4
0.6
0.4
CD8 lo / DC-LAMP lo (n = 25)
CD8 lo / DC-LAMP hi
or CD8 hi / DC-LAMP lo (n = 18)
CD8 hi / DC-LAMP hi (n = 9)
0.2
0.2
CD8 lo (n = 36)
CD8 hi (n = 16)
0
Time (months) 0
At risk
patients
B
0
20
40
60
80
100
Hi
16
7
4
2
0
0
Lo
36
27
16
11
6
4
Time (months) 0
120
Hi/Hi
At risk
patients
D
DC-LAMP+ mature DC:
60
80
100
0
0
0
0
18
13
7
5
2
1
25
19
13
8
5
4
120
NKp46+ NK cells:
P = 0.002
1
0.8
Overall survival
Overall survival
40
2
Lo/Lo
0.8
0.6
0.4
0.2
0.6
0.4
0.2
DC-LAMP lo (n = 32)
NKp46 hi (n = 28)
DC-LAMP hi (n = 20)
0
Time (months) 0
At risk
patients
20
MIX
P = 0.03
1
9
NKp46 lo (n =24)
0
20
40
60
80
100
Hi
20
10
5
3
1
0
Lo
32
24
16
10
6
5
120
Time (months) 0
At risk
patients
60
80
100
Hi
28
20
20
40
13
9
5
3
Lo
24
12
7
4
2
2
120
þ
þ
þ
Figure 3. Prognostic value of the densities of CD8 T cells, mature dendritic cells (DC-LAMP ), and NK cells (NKp46 ) in lung metastases from RCC.
Kaplan–Meier curves for the duration of OS according to a separated (A, B) and combined (C) analysis of CD8þ and DC-LAMPþ cell densities. D, Kaplan–Meier
curves for the duration of OS according to the densities of NKp46þ cells. The numbers of at risk patients according to a separated and combined
analysis of CD8þ and DC-LAMPþ densities and NKp46þ cells densities were given. Statistical comparison was conducted by the log-rank test and all
OS log-rank P values were corrected using the formula proposed by Altman and colleagues.
The density of DC-LAMPþ cells was manually counted
on the entire section as previously described (23). CD3þ,
CD8þ, granzyme Bþ, and NKp46þ cells were counted in
the center of the tumor and in the invasive margin of the
tumor with the convergence to the mean method (24).
For each slide, 40 to 100 high-power fields (1.37–3.43
mm2) were examined on each tumor zone. Both immunostaining and scoring were evaluated by 3 independent
observers blinded to clinical data (R. Remark, L. Crozet,
and A. Lupo, expert pathologist).
Gene expression analyses
RNA from the frozen tissues of 32 lung metastases
was extracted with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions and controlled for
quantity and quality on an Agilent 2100 Bioanalyser
(Agilent Technologies). Then, reverse transcription PCR
was conducted with the High-Capacity cDNA Reverse
4084
Clin Cancer Res; 19(15) August 1, 2013
Transcription kit (Applied Biosystem). Finally, the quantitative gene expression analysis of selected targets was
conducted in duplicates with the TaqMan Human
Immune Array on an Applied Biosystems 7900HT Fast
Real-Time PCR System. Expression levels of genes were
determined using threshold cycle (Ct) values normalized
to actin B (DCt) and were represented using the Genesis
program.
Statistical analyses
We used the Mann–Whitney test to compare the densities of infiltrating immune cells in the different tumors
and DCt, and the Wilcoxon matched pairs test to compare
the density of infiltrating immune cells in different
tumors from the same patient. Because all gene expression comparisons were preplanned and the 51 genes
clustered according to their immune functions before
analysis, the P values were not corrected by Bonferroni
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Published OnlineFirst June 19, 2013; DOI: 10.1158/1078-0432.CCR-12-3847
In Situ Immune Reaction in Lung Metastases
E
High
expression
–2.0
1:1
2.0
Low
expression
Mean(ΔCt) P = 0.0046
CD3E
CD4
CD8A
CD68
Immune cell
populations
Mean(ΔCt) P = 0.0094
IFNG
IL12A
IL12B
1L18
TBX21
LTA
TH1
orientation
Mean(ΔCt) P = 0.36
IL4
IL5
IL10
IL13
TH2
orientation
Mean(ΔCt) P = 0.16
C3
FN1
IL3
IL6
IL7
TNF
CSF1
IL1A
IL8
IL1B
IL17
PTGS2
SELE
CSF3
STAT3
Inflammation
Mean(ΔCt) P = 0.130
ACE
CD34
VEGF
Angiogenesis
Mean(ΔCt) P = 0.165
Immunosuppression
*
*
*
TGFB1
CTLA4
IL10
Mean(ΔCt) P = 0.126
GLNY
GZMB
PRF1
IL15
Cytotoxicity
Mean(ΔCt) P = 0.018
CCL2
CCL3
CCL5
CCR2
CCR5
Chemokines/
chemokine
receptors
CCL19
CCR4
CCR7
CXCL10
CXCL11
CXCR3
High CD8+/
DC-LAMP+
densities
Low CD8+/
DC-LAMP+
densities
Figure 3. (Continued ) E, expression of genes related to immune cell
populations, TH1/TH2 orientations, inflammation, angiogenesis,
immuno-suppression, cytotoxicity, chemokines/chemokine receptors
according to the CD8þ and DC-LAMPþ cell densities (high/high vs.
low/low) in lung metastases from RCC. Expression levels of genes were
determined using threshold cycle (Ct) values normalized to actin B
[ACTB] (DCt). We used Mann–Whitney test to identify genes with
significantly different levels of expression among patient groups (high vs.
low). , P < 0.05 for individual gene expression.
or similar methods. Correlations were evaluated by the
Spearman test. Overall survival (OS) curves were estimated by Kaplan–Meier method and differences between the
groups of patients were calculated using the log-rank test.
The start of follow-up for OS was the time of lung surgery.
In addition to mature dendritic cells, CD8þ T cells, and
NK cells densities, the following available clinical parameters were tested: initial stage (colorectal carcinoma),
completeness of resection at pulmonary level, number of
lung metastases, presence of extrathoracic metastases at
www.aacrjournals.org
time of lung surgery, thoracic lymph node invasion,
carcinoembryonic antigen (CEA) level (colorectal carcinoma), initial Fuhrman nuclear grade (RCC), presence
of metastases at presentation (RCC), time from lung
metastasis diagnosis to surgery (RCC), disease-free interval (RCC), alkaline phosphatase, hemoglobin, neutrophils, and platelets levels (RCC). The lower limit of
normal was used for hemoglobin (cutoff values: men
¼ 13 g/dL and women ¼ 12 g/dL) and the upper limit
(ULN) was used for alkaline phosphatase (cutoff value:
80 U/L), neutrophils (cutoff value: 7500/mm3), and
platelets (cutoff value: 400,000/mm3). With respect to
immune cell densities and number of metastases, the
"minimum P value" approach was used to determine the
cutoff for the best separation of patients referring to their
OS outcome (outcome-oriented approach). Because the
P values obtained might be overestimated, OS log-rank
P values were corrected using the formula proposed by
Altman and colleagues (25) and using 10-fold crossvalidations as recommended by Faraggi and colleagues
(26). The confidence interval was important around
the optimal P value (Supplementary Table S3). We have
also ensured that the significance established at the
optimal cutoff remained valid at the quartiles (dataoriented approach). A P value less than 0.05 was considered statistically significant. Independent parameters
identified at univariate analysis as possibly influencing
outcome (P < 0.1) were introduced in a multivariate Coxproportional hazards regression model. All analyses were
conducted with Prism 5 (GraphPad), Statview (Abacus
Systems), and the R (http://www.r-project.org/).
Results
The densities of immune cells correlate with OS in lung
metastases from colorectal carcinoma
Because densities of CD8þ T cells and DC-LAMPþ mature
dendritic cells in primary tumors correlate with survival (1),
we counted these cells in lung metastases from 140 colorectal carcinoma patients. We also quantified NKp46þ NK
cells as a marker of innate immune response. High densities
of infiltrating CD8þ T cells (Fig. 1A) and mature dendritic
cells (Fig. 1B) were associated with prolonged OS (P ¼ 0.039
and 0.001, respectively). Combination of these 2 immune
parameters allowed to identify patients with better outcome
(CD8high/DC-LAMPhigh; Fig. 1C, P ¼ 0.008). NKp46þ cell
density did not predict clinical outcome (P ¼ 0.12; Fig. 1d).
Significance was established at the optimal cutoff, but
remained valid at quartiles including the median (Supplementary Table S3). The quantification of CD8þ T cells
separately in the center of the tumor and the invasive margin
regions yielded similar results (Supplementary Fig. S1).
Univariate analysis of other clinical and pathological parameters is reported in Table 1. At multivariate analysis (Table
1), immune pattern (CD8þ/DC-LAMPþ densities) of metastases was the strongest independent predictor of survival.
As reported in colorectal carcinoma primary tumors (3),
gene expression analyses revealed that a strong CD8þ and
DC-LAMPþ cell infiltration was associated with a higher
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Remark et al.
expression of genes linked to TH1 orientation, cytotoxicity,
and lymphoid chemokines/chemokine receptors in lung
metastases (Fig. 1e). Expressions of clusters of genes associated with TH2 orientation, inflammation, angiogenesis,
or immunosuppression were not correlated with the CD8þ/
DC-LAMPþ densities. However, individual gene expression
of VEGF was inversely correlated with CD8þ/DC-LAMPþ
infiltration, as reported in primary colorectal carcinoma
(3, 27) whereas that of IL17 and CTLA4 were positively
correlated (Fig. 1e).
The in situ immune pattern is reproduced from primary
tumors to metastases in colorectal carcinoma
To investigate whether the in situ immune pattern varies
during the course of the metastatic disease for a given
patient, we analyzed coincident colorectal carcinoma lung
metastases occurring in the other lung side (n ¼ 14) operated 1 to 9 months after the initial metastatic surgery, and/or
relapsing metastasis occurring 14 to 52 months after surgical removal of the lung metastasis (n ¼ 12; Fig. 2A).
Densities of CD8þ (Fig. 2B), DC-LAMPþ (Fig. 2C), and
NKp46þ (Fig. 2D) cells were not significantly different
between 2 coincident metastatic sites or between the first
lung metastasis and its relapse. We found correlations in the
densities of immune cells between coincident and relapsing
metastases (Fig. 2B–D).
To address the question of the relationship between
immune cell densities in the primary tumor and metastasis,
we compared immune infiltrates of primary tumors and
lung metastases from the same individuals (n ¼ 25; Fig.
2E). Primary colorectal carcinoma differed from lung
metastases by significantly higher density of CD8þ T cells
(P < 0.05; Fig. 2F), but the density of each cell type was
positively correlated between the primary and the metastatic tumors (Fig. 2F–H for CD8þ, DC-LAMPþ, and NKp46þ
cells, respectively). We had access to a small number (n ¼ 5)
of matched hepatic metastases and the correlation was also
found between primary colorectal carcinoma, lung, and
liver metastases (data not shown).
The densities of immune cells correlate with OS in lung
metastases from RCC
We have also analyzed a cohort of 52 RCC lung metastases. Patients with high densities of infiltrating CD8þ T cells
(Fig. 3A) or DC-LAMPþ cells (Fig. 3B) have reduced survival
(P ¼ 0.03). These 2 immune parameters allowed to identify,
with strong significance, patients with poorer outcome
(CD8high/DC-LAMPhigh; Fig. 3C, P < 0.0001). High density
of NKp46þ cells was associated with improved survival (P ¼
0.002; Fig. 3D). Separate analysis of the CD8þ and NKp46þ
immune infiltrates in the center of the tumor and invasive
margin also correlated with OS (Supplementary Fig. S2).
Significance was established at the optimal cutoff but also
conserved at the quartiles (Supplementary Table S3). Univariate proportional hazard Cox analyses revealed that the
immune pattern (CD8þ/DC-LAMPþ densities), NKp46þ cell
density, presence of metastases at presentation, and diseasefree interval were the only prognostic factors of patients’
4086
Clin Cancer Res; 19(15) August 1, 2013
survival in our cohort (Table 1). Our data also suggest that
hemoglobin and thoracic lymph node invasion tended to be
associated with survival (P ¼ 0.061 and 0.086, respectively).
In the resulting multivariate proportional hazard Cox model, DFI and immune pattern were independent prognostic
factors (P ¼ 0.0067 and 0.0039, respectively; Table 1).
A strong CD8þ/DC-LAMPþ infiltration was associated
with a higher expression of genes linked to TH1 orientation,
lymphoid, and myeloid chemokine/chemokine receptors.
Contrasting with colorectal carcinoma, cytotoxicity-related
genes were highly expressed in both groups of tumors
(refs. 3, 27; Fig. 3E) and, interestingly, VEGF gene expression was positively correlated with CD8þ/DC-LAMPþ infiltration, as well as that of interleukin-6 and STAT3.
As previously shown in colorectal carcinoma, we found a
correlation between the density of infiltrating DC-LAMPþ,
CD8þ, and NKp46þ cells in the primary tumor and in the
corresponding lung metastasis (n ¼ 24; Fig. 4A–D), indicating that the in situ immune pattern of the primary tumor
was reproduced in the metastasis.
The cell composition, organization, and polarization
of the immune reaction is different in colorectal
carcinoma and RCC lung metastases
Because CD8þ, DC-LAMPþ, and NKp46þ cell densities in
lung metastases have different clinical impacts in colorectal
carcinoma and RCC, we compared their microenvironments. Histologic analyses revealed profound differences
between colorectal carcinoma and RCC lung metastases. We
found glands, often necrotic, in an abundant and collagenous stroma surrounded by a high density of tertiary lymphoid structures (TLS) in colorectal carcinoma metastases
(Fig. 5A). In contrast, in RCC metastases, tumor cell nests
were separated by a thin stroma with few and scattered TLS
(Fig. 5A). TLS contained a B-cell follicle, a T-cell zone, and
PNAdþ high-endothelial venules (Fig. 5B).
We found similar densities of CD3þ and CD8þ T cells in
the whole tumor zone (Fig. 5C). Mature dendritic cells,
located in the T-cell area of TLS, were found at higher density
in colorectal carcinoma than in RCC (P < 0.0001; Fig. 5B
and C), in accordance with the higher number of TLS in
colorectal carcinoma lung metastases. The colorectal carcinoma metastases contained significantly lower densities of
NK cells as compared to RCC metastases (P < 0.0001; Fig. 5B
and C). No significant differences in the densities of CD8þ,
DC-LAMPþ, and NKp46þ cells were observed in tumors
from colorectal carcinoma or RCC patients having received
or not preoperative treatment (chemotherapy, IL-2/IFN, or
association of bevacizumab and chemotherapy; Fig. S3 and
Supplementary Tables S1 and S2 for treatment details).
Although expression of genes linked to adaptive immune
populations was not significantly different between both
types of metastatic tumors, we found a lower expression
of CD68 gene in colorectal carcinoma lung metastases
(Fig. 5D and Supplementary Fig. S4 for detailed gene
level expression). A similar TH1 orientation was found in
colorectal carcinoma and RCC metastases, but a stronger
expression of genes linked to TH2 was detected in RCC
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Published OnlineFirst June 19, 2013; DOI: 10.1158/1078-0432.CCR-12-3847
In Situ Immune Reaction in Lung Metastases
Primary tumor versus lung metastasis:
A
C
R = 0.689
ns
8,000
6,000
4,000
2,000
D
R = 0.547
*
15
Number of DC-LAMP+ cells/mm2
10,000
10
5
RC
T
M
L
C-
RC
lung metastases. Genes linked to acute inflammation were
upregulated in colorectal carcinoma lung metastases and
genes linked to chronic inflammation, angiogenesis, or
immunosuppression were upregulated in RCC lung metastases. A higher expression of cytotoxicity-related genes in
RCC metastases was observed, in accordance with their
higher NK-cell content. Chemokines and receptors genes
prone to attract TH1, T regulatory, and dendritic cells were
more expressed in colorectal carcinoma metastases, whereas
RCC lung metastases were characterized by the expression
of inflammatory chemokines and chemokine receptors
genes.
Discussion
The objective of our study was to characterize the immune
microenvironment of metastatic lesions and its clinical
impact. If several clinical parameters have been reported to
be associated with survival in metastatic patients, none has
obtained general agreement (17, 28), justifying the search of
new nonclinical prognostic markers. We report here a major
prognostic value of the immune pattern (densities of mature
dendritic cells and CD8þ T cells) in metastases from colorectal carcinoma and RCC, although with opposite impact
on OS. In our cohorts of oligometastatic surgically treated
600
400
200
0
0
T
P
C-
R = 0.817
ns
800
Number of NKp46+ cells/mm2
B
0
www.aacrjournals.org
Lung
metastasis
Primary
tumor
Number of CD8+ cells/mm2
þ
þ
Figure 4. CD8 T cells, DC-LAMP
mature dendritic cells, and NKp46þ
NK cell densities in metastases and
in primary RCC tumors. A, surgical
treatment for primary RCC and
their lung metastases. B–D, RCC
primary tumors were less infiltrated
by DC-LAMPþ cells than lung
metastases. R values show the
positive correlations (0.5 < R < 0.9
and P < 0.05, Spearman test)
between primary tumors and
lung metastases according to the
CD8þ, DC-LAMPþ, and NKp46þ
cell densities. PT, primary tumor;
LM, lung metastasis; ns, not
significant; , P < 0.05 (Wilcoxon
matched pairs test).
P
C-
RC
PT
M
RC
L
C-
C-
RC
M
-L
C
RC
patients, the strongest prognosticator was the immune
pattern, that is CD8þ and DC-LAMPþ cell density combination, as reported for many primary tumors (1–13, 23,
27, 29–31). NK cells density had also a prognostic value in
RCC. It seems that the immune pattern is a powerful
prognostic factor and a potentially important parameter
for metastatic patients’ management. Because of the incomplete data collection (especially for laboratory values which
were difficult to collect in a retrospective analysis), conclusions remain difficult to draw on the prognostic value of the
Memorial Sloan-Kettering Cancer Center (32) and Heng
and colleagues (33) prognostic factor models. Our previous
studies showed the highly clinical impact of the CD8þ cell
densities in primary colorectal carcinoma up to stage III,
that is without distant metastases at the time of diagnosis
(2). In this study, the impact of the immune pattern on OS
was lower in primary tumors (P ¼ 0.15 and 0.01 for CD8þ
and DC-LAMPþ cells, respectively; data not shown) than in
lung metastases from colorectal carcinoma (P ¼ 0.039 and
0.001 for CD8þ and DC-LAMPþ cells, respectively; data not
shown).
Clinical significance of CD8þ T cells density seems to be
contrasted, according to primary tumor’s origin. Although
we found similar densities of CD8þ T cells in both colorectal
carcinoma and RCC metastases, the prognostic value of
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Remark et al.
A
B
Colorectal cancer Lung metastasis
Renal cell carcinoma Lung metastasis
Colorectal cancer Lung metastasis
Renal cell carcinoma Lung metastasis
Figure 5. Comparison of the immune contextures in colorectal carcinoma
and RCC lung metastases. A, representative pictures of colorectal
carcinoma and RCC lung metastases [hematoxylin–eosin–safran (HES)
4088
Clin Cancer Res; 19(15) August 1, 2013
these cells was different. Similar conflicting observations
about the prognostic role of immune infiltrate have been
reported in primary colorectal carcinoma and RCC
(2, 4, 15, 27) and one could hypothesize that the seeding
organ (colon or kidney) may explain this variability in the
outcome. Because it remains valid in the lung metastases,
our data support the idea that the kind of primary tumor is
essential in determining the prognostic value of the host
immune infiltrate at metastatic level. This is in accordance
with the fact that primary RCC seems as an exception to the
well-documented general findings that TH1/CD8 immune
cell infiltrate and high density of mature dendritic cells
correlate with favorable prognosis in the majority of solid
tumors (1). The differential clinical impacts of the T cells
might be due to their site of activation. Indeed, we have
previously reported that TLS in early stages of non–small
cell lung cancer may act as potential structures of antitumor
T-cell generation (23, 34). We found more TLS, reflected by
higher densities of mature dendritic cells and higher expression of CCL19 gene, a chemokine expressed in TLS (34), in
colorectal carcinoma than in RCC metastases. Because TLS
are scarce in RCC lung metastases and numerous in lung
metastases from colorectal carcinoma, one may postulate
that the T cells present in the former have not been educated
in tumor-adjacent TLS (35) and reflect rather a chronic
inflammatory reaction which is known to be deleterious
for the host (36). Indeed, gene expression analyses revealed
significant differences between lung metastases from colorectal carcinoma and RCC, which share a TH1 profile, but
the latter exhibit also a TH2, inflammatory, and immunosuppressive pattern. The high expression of VEGF, IL-6, and
M-CSF genes in RCC may also inhibit the differentiation of
dendritic cells and induce monocyte differentiation to
macrophages (37–39), which could initiate an impaired
T-cell response in RCC, resulting in poor prognosis. Interestingly, VEGF gene expression was positively correlated
with high CD8þ and DC-LAMPþ infiltration in RCC lung
metastases and with low CD8þ and DC-LAMPþ infiltration in colorectal carcinoma lung metastases. Because it
has been suggested that VEGF may induce non-coordinated immune responses (27), affect cytotoxic TH1 adaptive immune responses (39, 40) and contribute to the
progression of malignant disease, the correlation between
CD8þ/DC-LAMPþ densities and VEGF expression could
be one explanation among others to explain the negative
impact associated with this immune signature. Moreover,
upregulation of IL6 and STAT3 genes in the CD8high/DCLAMPhigh group could reflect the inflammatory milieu of
the RCC microenvironment (41, 42). It could also explain
the reasons that immunotherapies, which modify the
staining] showing the organization of tumors. Original magnification: 40
and 200. TLS, tertiary lymphoid structure; T, tumor; S, stroma. B,
þ
location and organization of CD20 B-cell follicles (red) surrounded by
high-endothelial venules (blue), DC-LAMP expressing mature dendritic
cells (red, black arrows), CD3þ T cells (red), CD8þ T cells (red), and
NKp46þ NK cells (red) in colorectal carcinoma (left) and RCC (right) lung
metastases. Original magnification: 200 and 400.
Clinical Cancer Research
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Published OnlineFirst June 19, 2013; DOI: 10.1158/1078-0432.CCR-12-3847
In Situ Immune Reaction in Lung Metastases
C
CD8+ T cells:
CD3+ T cells:
ns
10,000
D
–2.0
High
expression
ns
10,000
1:1
2.0
Low
expression
Number of CD8+ cells/mm2
Number of CD3+ cells/mm2
CD68
1,000
CD3E
CD4
CD8A
Th1
orientation
100
Th2
orientation
LM
LM
C-
C-
DC-LAMP+ mature
***
CR
M
M
L
C-
L
C-
RC
ns
IL18
TBX21
IFNG
IL12A
IL12B
LTA
ns
IL5
IL4
*
IL10
IL13
ns
FN1
C3
VEGF
STAT3
CSF1
ACE
CD34
IL6
IL7
TNF
IL3
*
IL8
IL1A
***
1,000
Number of NKp46+ cells/mm2
0.01
Inflammation
and
angiogenesis
NKp46+ NK cells:
10
0.1
C-
RC
CR
1
LM
LM
C-
RC
CR
*
1,000
10
100
Number of DC-LAMP+ cells/mm2
Immune cell
populations
Immunosuppression
*
IL1B
PTGS2
SELE
CSF3
IL17
ns
TGFB1
CSF1
*
CTLA4
IL10
ns
GZMB
GLNY
PRF1
IL15
*
100
Cytotoxicity
CCR2
CCL2
CCR5
CCL3
CCL5
10
Chemokines/
chemokines
receptors
1
M
M
CR
L
C-
L
C-
RC
Colorectal
carcinoma - LM
*
CCR4
CCL19
CXCL11
*
CXCR3
CXCL10
CCR7
ns
Renal cell
carcinoma - LM
þ
þ
þ
þ
Figure 5. (Continued ) C, quantification of CD3 , CD8 , DC-LAMP , and NKp46 cells in lung metastases from colorectal (colorectal carcinoma-LM,
n ¼ 140) and renal cell carcinoma (RCC-LM, n ¼ 52). Whiskers length represents 10 to 90 percentile. ns, not significant; , P < 0.0001 (Mann–Whitney test).
D, heat map of the expression levels of genes according to the origin of lung metastases (colorectal carcinoma and RCC) represented using the Genesis
program. LM, lung metastasis; ns, not significant; , P < 0.05 (Mann–Whitney test).
acute/chronic inflammatory microenvironment, are often
reported to have some efficacy in metastatic renal cell
carcinoma (43).
The Von Hippel Lindau phenotype, often found in RCC,
may also be involved in the shaping of peculiar tumor
microenvironments, through induction of hypoxia, production of VEGF, induction of regulatory immune circuits
(44–47), and increased sensitivity of tumor cells to NK-cell
lysis (48). It may also influence differently the stroma
characteristics, the vascularization, or the collagen content
which could also impact on the migration, organization,
and functionality of intratumor immune cells (49). Together, these data may explain the negative clinical impact of the
adaptive immune pattern at the primary and advanced
stages of RCC.
www.aacrjournals.org
We found that colorectal carcinoma and RCC have a
correlated pattern of DC-LAMPþ, CD8þ, and NKp46þ cells,
from primary tumor to relapsing metastasis, which could
reflect, either a potential "imprinting" of the immune
microenvironment by the tumor cells or the possibility that
the immune contexture in the primary tumor, results in
"educated" immune cells that are recalled in the metastatic
sites.
In conclusion, our findings highlight the fact that during all
steps of cancer development, reciprocal interactions occur
between immune and cancer cell and are critical for patients’
survival. The immune signature seems to be a phenotypic
marker for the disease and is remarkably reproduced between
primary and metastatic sites in the same patient. The immune
contexture affects OS in lung metastases from colorectal
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4089
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Remark et al.
carcinoma and RCC, and the analysis of the immune pattern
might be useful to guide therapeutics (50).
Disclosure of Potential Conflicts of Interest
S. Oudard has honoraria from speakers bureau of Sanofi, Roche, Novartis,
BMS, Takeda, and Jansen. No potential conflicts of interest were disclosed by
the other authors.
Authors' Contributions
Conception and design: R. Remark, J.-F. Regnard, C. Sautes-Fridman, W.H.
Fridman, D. Damotte
Development of methodology: R. Remark, M.-C. Dieu-Nosjean, D.
Damotte
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): R. Remark, M. Alifano, A. Lupo, M. Riquet, H.
Ouakrim, J. Goc, A. Cazes, J.F. Flejou, L. Gibault, J.-F. Regnard, O.N. Pages, S.
Oudard, D. Damotte
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Remark, M. Alifano, I. Cremer, M.-C.
Dieu-Nosjean, S. Oudard, W.H. Fridman, D. Damotte
Writing, review, and/or revision of the manuscript: R. Remark, M.
Alifano, I. Cremer, M.-C. Dieu-Nosjean, J. Goc, S. Oudard, C. Sautes-Fridman, W.H. Fridman, D. Damotte
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. Remark, L. Crozet, A. Cazes,
L. Gibault, V. Verkarre, B. Mlecnik, C. Sautes-Fridman, W.H. Fridman,
D. Damotte
Study supervision: W.H. Fridman, D. Damotte
Acknowledgments
The authors thank P. Bonjour, V. Ducruit, T. Fredriksen for technical
assistance and M. Bovet for help in clinical data collection, the H^
otel-Dieu
hospital tumor bank (no. DC 2009-947), the tumorotheque cancer-est
(Tumo0203), and the "Centre d’Imagerie Cellulaire et de Cytometrie"
(Cordeliers Research Center, Paris).
Grant Support
This work was supported by Institut National de la Sante et de la
Recherche Medicale (INSERM), Universite Paris-Descartes, Universite Pierre
et Marie Curie, Institut National du Cancer, Canceropole Ile de France, and
Labex Immuno-oncology (2011-1-PLBIO-06-INSERM 6-1, PLBIO09-088IDF-KROEMER, 11LAXE62_9UMS872 FRIDMAN).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received December 21, 2012; revised May 23, 2013; accepted June 7, 2013;
published OnlineFirst June 19, 2013.
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