Published OnlineFirst June 19, 2013; DOI: 10.1158/1078-0432.

CCR-12-3847

Clinical
Cancer
Human Cancer Biology Research

Characteristics and Clinical Impacts of the Immune

Environments in Colorectal and Renal Cell Carcinoma Lung
Metastases: Inuence 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,
Aure
jou2,8, Laure Gibault3,9, Virginie Verkarre3,10, Jean-Francois Re

lie Cazes3,6, Jean-Francois Fle
gnard3,5,
 5

3,6
Olivier-Nicolas Pages , Stephane Oudard , Bernhard Mlecnik 1,2,3

, Catherine Sautes-Fridman 1,2,3
,
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 192) and qPCR (n 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-LAMP mature dendritic cells (P < 0.0001) and lower densities of NKp46 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 CD8 and DC-LAMP cells correlated with longer overall survival (OS) in
colorectal carcinoma (P 0.008) and shorter OS in RCC (P < 0.0001). High NK-cell densities were
associated with improved survival in RCC (P 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 (CD8/DC-LAMP 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); 407991. 2013 AACR.

Authors' Afliations: 1Institut National de la Sante
et de la Recherche

Introduction
Me
dicale (INSERM), U872, Centre de Recherche des Cordeliers; 2Uni- Immune cells are found in human solid tumors, and the

Pierre et Marie Curie-Paris 6, UMRS 872; Universite
versite 3

Paris Des-
cartes-Paris 5, UMRS 872; 4Universite
Denis Diderot-Paris 7; 5Services
immune pattern of the tumor microenvironment is a major
d'anatomie-pathologique et de chirurgie thoracique, Ho ^ pital Ho
^ tel Dieu; predictor of patient survival in a large array of primary
6
Services d'anatomie-pathologique, oncologie et de chirurgie thoracique; tumors (1). Thus, a high density of T cells with a TH1 and
7
^ pital Europe

en Georges Pompidou;
8
Service d'Immunologie Biologique, Ho
Service d'anatomie-pathologique, Ho ^ pital Saint-Antoine; 9Service d'ana- CD8 T cells cytotoxic orientation or of mature dendritic
tomie-pathologique, Ho ^ pital Cochin; and Service d'anatomie-patholo-
10 cells (DC) are beneficial in most cancers, especially in
gique, Ho^ pital Necker-Enfants Malades, AP-HP, Paris, France colorectal (24), lung (5), breast (6), gastric (7), pancreatic
Note: Supplementary data for this article are available at Clinical Cancer (8), urothelial (9), hepatocellular (10), esophageal (11),
Research Online (http://clincancerres.aacrjournals.org/). ovarian cancer (12) and melanoma (13), with the exception
W.-H. Fridman and D. Damotte contributed equally to this article.
of renal cell carcinoma (RCC) in which high densities of
CD8 and CD45RO cells are associated with poor prog-
Corresponding Author: Diane Damotte, INSERM U872, Centre de

decine, 75006 Paris,
Recherche des Cordeliers, 15 rue de l'Ecole de Me nosis (14, 15).
France. Phone: 33-1-42-34-87-12; Fax: 33-1-42-34-86-41; E-mail: Even if metastatic spreading is the main cause of death
diane.damotte@htd.aphp.fr by cancer (16), metastatic patients have heterogeneous
doi: 10.1158/1078-0432.CCR-12-3847 survival (17). A classical view of cancer progression is that
2013 American Association for Cancer Research. genetic modifications (18) may allow malignant cells to

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Remark et al.

escape local and systemic immune control (19) and

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 CD8 T cells and DC-LAMP mature
dendritic cells ("immune pattern"), evaluated in paraf-
fin sections, were independent prognostic factors of
patients survival, and stronger prognosticators than
currently evaluated clinical and pathological para-
meters. 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 envi-
ronment and the patients outcome.
consequently invade and metastasize in distant organs.
This hypothesis would predict that the immune micro-
environment 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
CD8 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 colo-
rectal carcinoma and RCC metastases seeded in a same
organ, the lung, compared coincident and relapsing

A CD8+ T cells: C CD8+ and DC-LAMP+ cells:

1 P = 0.039 1 P = 0.008

0.8 Overall survival 0.8

Overall survival

0.6 0.6

0.4 0.4

CD8 hi / DC-LAMP hi (n = 67)

0.2 0.2 CD8 lo / DC-LAMP hi
CD8 hi (n = 71)
or CD8 hi / DC-LAMP lo (n = 53)
CD8 lo (n = 69)
0 0 CD8 lo / DC-LAMP lo (n = 20)

Time (months) 0 20 40 60 80 100 120 Time (months) 0 20 40 60 80 100 120

Hi 71 53 40 24 12 4 Hi/Hi 67 51 39 24 13 4
At risk
patients At risk MIX 53 42 24 9 3 1
Lo 69 52 30 15 5 2
patients
Lo/Lo 20 12 7 6 2 1

B DC-LAMP+ mature DC: D NKp46+ NK cells:

1 P = 0.001 1 P = 0.12

0.8 0.8
Overall survival
Overall survival

0.6 0.6

0.4 0.4

0.2 0.2
DC-LAMP hi (n = 116) NKp46 hi (n = 58)
DC-LAMP lo (n = 24)
0 0 NKp46 lo (n = 26)

Time (months) 0 20 40 60 80 100 120 Time (months) 0 20 40 60 80 100 120

Hi 116 91 62 33 16 5 At risk Hi 58 49 39 23 14 5
At risk
patients Lo 24 14 8 6 2 1 patients Lo 26 17 8 3 0 0

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. KaplanMeier curves for the duration of OS according to a separated (A, B) and combined (C) analysis of CD8 and DC-LAMP densities
in colorectal carcinoma lung metastases. D, KaplanMeier curves for the duration of OS according to the densities of NKp46 cells in colorectal
carcinoma lung metastases (n 84). The numbers of at risk patients according to a separated and combined analysis of CD8 and DC-LAMP densities and
NKp46 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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In Situ Immune Reaction in Lung Metastases

E High Low expression

expression
2.0 1:1 2.0
Mean(Ct) P = 0.014
CD3E
Immune cell CD4
CD8A
populations CD68

Mean(Ct) P = 0.04
IFNG
IL12A
TH1 IL12B
orientation 1L18
TBX21
LTA
Mean(Ct) P = 0.42
IL4
TH2 IL5
Figure 1. (Continued ) E, expression IL10
orientation IL13
of genes related to immune cell
populations, TH1/TH2 orientations, Mean(Ct) P = 0.39
inammation, angiogenesis, C3
immuno-suppression, cytotoxicity, FN1
IL3
chemokines/chemokine receptors IL6
according to the densities of CD8 IL7
TNF
and DC-LAMP cells (high/high vs. CSF1
IL1A
low/low) in lung metastases from IL8
Inflammation IL1B
colorectal carcinoma. Expression
levels of genes were determined
IL17
PTGS2 *
SELE
using threshold cycle (Ct) values CSF3
normalized to actin B [ACTB] (DCt). STAT3

We used MannWhitney test to mean(Ct) P = 0.13

identify genes with signicantly ACE
different levels of expression Angiogenesis CD34
VEGF
among patient groups (high vs. low *
CD8/DC-LAMP densities). mean(Ct) P = 0.23

, P < 0.05 for individual gene Immuno- TGFB1
expression. suppression
CTLA4
IL10 *
mean(Ct) P = 0.032
GLNY
GZMB
Cytotoxicity PRF1
IL15

mean(Ct) P = 0.03
CCL2
CCL3
CCL5
CCR2
Chemokines/ CCR5
chemokine CCL19
CCR4
receptors CCR7
CXCL10
CXCL11
CXCR3

High CD8+/DC-LAMP+ Low CD8+/DC-LAMP+

densities 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-LAMP mature
dendritic cells and CD8 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
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 Multivariate analyses

Variable HR 95% CI P value HR 95% CI P value

Colorectal carcinoma Stage (stage 3 4 vs. stage 1 2)
a
1.68 (0.883.20) 0.116 Not included in the
lung metastases Presence of extrathoracic metastases 1.56 (0.882.75) 0.128 multivariate analysis
(yes vs. no)
Completeness of resection (R1 vs. R0) 2.49 (0.778.05) 0.129
CEA level (5 ng/mL vs. <5 ng/mL) 1.55 (0.862.82) 0.148
NK cells (high vs. low) 0.58 (0.281.16) 0.123
Thoracic lymph node invasion (yes vs. no) 1.49 (0.603.66) 0.0503 1.80 (0.873.72) 0.115
Number of metastases (>2 vs. 2) 1.84 (1.033.28) 0.0405 2.17 (1.203.93) 0.011
Immune pattern (high/mix/low) 0.54 (0.390.76) 0.0002 0.54 (0.390.75) 0.0003
RCC lung metastases Initial Fuhrman nuclear grade (3 4 vs. 1.32 (0.563.11) 0.533 Not included in the
1 2) multivariate analysis
Time from lung metastasis diagnosis to 1.77 (0.833.76) 0.142
surgery (>1 year vs. 1year)
Number of metastases (multiple vs. 1) 1.30 (0.612.79) 0.501
Presence of extrathoracic metastases 1.34 (0.513.55) 0.555
(yes vs. no)
Completeness of resection (R1 vs. R0) 1.40 (0.424.65) 0.587
Alkaline phosphatase (>80 U/L vs. 80 U/L) 1.52 (0.754.33) 0.682
Neutrophils (>7,500/mm3 vs. 7,500/mm3) 0.87 (0.292.62) 0.805
Platelets (>400,000/mm3 vs. 400,000/mm3) 0.80 (0.232.82) 0.728
DFI (1 year vs. <1 year) 0.35 (0.160.74) 0.0064 0.33 (0.140.73) 0.0067
Metastases at presentation (synchronous 2.23 (1.024.86) 0.0435 1.74 (0.476.46) 0.407
vs. metachronous)
Thoracic lymph node invasion (yes vs. no) 1.92 (0.914.05) 0.086 2.41 (0.817.17) 0.113
Hemoglobin (men: <13 g/dL vs. 13 g/dL 2.26 (0.965.33) 0.061 1.00 (0.342.67) 0.935
and women: <12 g/dL vs. 12 g/dL)
NK cells (high vs. low) 0.46 (0.220.95) 0.037 0.32 (0.141.03) 0.0579
Immune pattern (high/mix/low) 2.68 (1.584.57) 0.00028 2.70 (1.375.29) 0.0039

NOTE: To be able to conduct regression with a categorical variable, they were coded before entered into the Cox model. Parameters
with signicant 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 Europ
een Georges Pompi-
dou 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 car-
cinoma lung metastases from the same patients were stud-
ied. 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 car-
cinoma (n 19) or RCC (n 13).
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 pathol-
ogists) 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, Spring-
bioscience), CD20 (L26, Dako), DC-LAMP (1010.01, Den-
dritics), granzyme B (11F1, Novocastra), NKp46 (195314,

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In Situ Immune Reaction in Lung Metastases

A Coincident metastases: Relapsing metastases:

t = 19 t = 1452
months months

First Second First

side metastasis Metastasis
side relapse

B R = 0.644 R = 0.643 C R = 0.614 R = 0.580 D R = 0.696

ns
R = 0.895
ns
ns ns ns ns
4,000 15 400
Number of DC-LAMP+ cells/mm2

Number of NKp46+ cells/mm2

Number of CD8+ cells/mm2

3,000 300
10

2,000 200

5
1,000 100

0 0 0
e e is se e e is s e e e is e
id sid ta
s
ap id sid ta
s
ap id sid as ps
ts d s re
l ts d s re
l ts d st re
la
rs on et
a rs on et
a rs on et
a
Fi c is Fi c is Fi c is
Se tm as Se tm as Se tm as
rs st rs st rs st
Fi et
a Fi et
a Fi et
a
M M M

E F R = 0.693 G R = 0.659 H R = 0.656

Primary tumor versus metastases: ns ns
10,000 * 15 800
Number of DC-LAMP+ cells/mm2

Number of NKp46+ cells/mm2

Number of CD8+ cells/mm2

8,000
600
10
6,000
400
4,000
Primary Lung 5
tumor metastasis
200
2,000

0 0 0
T M T M PT
-P -L -P -L LM
C C C C C- C-
CR CR CR CR CR CR

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. BD, coincident or relapsing metastases have the
same densities of CD8, DC-LAMP, and NKp46 cells. E, surgical treatment for primary colorectal carcinoma and their lung metastases. FH,
colorectal carcinoma primary tumors were more inltrated by CD8 cells than lung metastases, but have similar densities of DC-LAMP and NKp46 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 CD8, DC-LAMP, and NKp46 cell densities. PT, primary tumor; LM, lung metastasis; ns, not signicant;

, P < 0.05 (Wilcoxon matched pairs test).

R&D Systems), or PNAd (MECA-79, BD Pharmingen)] was revealed using 3-amino-9-ethylcarbazole substrate
followed by secondary antibodies coupled with biotin (Vector Laboratories). Alkaline phosphatase activity was
or alkaline phosphatase. Biotinylated antibodies were cou- revealed using alkaline phosphatase substrate III (Vector
pled with streptavidin-peroxidase and peroxidase activity Laboratories).

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Remark et al.

A CD8+ T cells: C CD8+ and DC-LAMP+ cells:

1 P = 0.03 1 P < 0.0001

0.8 0.8

Overall survival
Overall survival

0.6 0.6

0.4 0.4

CD8 lo / DC-LAMP lo (n = 25)

0.2 0.2
CD8 lo / DC-LAMP hi
CD8 lo (n = 36)
or CD8 hi / DC-LAMP lo (n = 18)
0 CD8 hi (n = 16) 0 CD8 hi / DC-LAMP hi (n = 9)

Time (months) 0 20 40 60 80 100 120 Time (months) 0 20 40 60 80 100 120

Hi 16 7 4 2 0 0 Hi/Hi 9 2 0 0 0 0
At risk
patients At risk MIX 18 13 7 5 2 1
Lo 36 27 16 11 6 4
patients
Lo/Lo 25 19 13 8 5 4

B DC-LAMP+ mature DC: D NKp46+ NK cells:

1 P = 0.03 1 P = 0.002

0.8 0.8
Overall survival

0.6 Overall survival 0.6

0.4 0.4

0.2 0.2
DC-LAMP lo (n = 32) NKp46 hi (n = 28)
0 DC-LAMP hi (n = 20) 0 NKp46 lo (n =24)

Time (months) 0 20 40 60 80 100 120 Time (months) 0 20 40 60 80 100 120

At risk Hi 20 10 5 3 1 0 Hi 28 20 13 9 5 3
At risk
patients Lo 32 24 16 10 6 5 patients Lo 24 12 7 4 2 2

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.
KaplanMeier curves for the duration of OS according to a separated (A, B) and combined (C) analysis of CD8 and DC-LAMP cell densities. D, KaplanMeier
curves for the duration of OS according to the densities of NKp46 cells. The numbers of at risk patients according to a separated and combined
analysis of CD8 and DC-LAMP densities and NKp46 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-LAMP cells was manually counted Transcription kit (Applied Biosystem). Finally, the quan-
on the entire section as previously described (23). CD3, titative gene expression analysis of selected targets was
CD8, granzyme B, and NKp46 cells were counted in conducted in duplicates with the TaqMan Human
the center of the tumor and in the invasive margin of the Immune Array on an Applied Biosystems 7900HT Fast
tumor with the convergence to the mean method (24). Real-Time PCR System. Expression levels of genes were
For each slide, 40 to 100 high-power fields (1.373.43 determined using threshold cycle (Ct) values normalized
mm2) were examined on each tumor zone. Both immu- to actin B (DCt) and were represented using the Genesis
nostaining and scoring were evaluated by 3 independent program.
observers blinded to clinical data (R. Remark, L. Crozet,
and A. Lupo, expert pathologist). Statistical analyses
We used the MannWhitney test to compare the den-
Gene expression analyses sities of infiltrating immune cells in the different tumors
RNA from the frozen tissues of 32 lung metastases and DCt, and the Wilcoxon matched pairs test to compare
was extracted with the RNeasy Mini Kit (Qiagen) accord- the density of infiltrating immune cells in different
ing to the manufacturers instructions and controlled for tumors from the same patient. Because all gene expres-
quantity and quality on an Agilent 2100 Bioanalyser sion comparisons were preplanned and the 51 genes
(Agilent Technologies). Then, reverse transcription PCR clustered according to their immune functions before
was conducted with the High-Capacity cDNA Reverse 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

time of lung surgery, thoracic lymph node invasion,

E High
expression
Low
expression carcinoembryonic antigen (CEA) level (colorectal carci-
2.0 1:1 2.0
noma), initial Fuhrman nuclear grade (RCC), presence
Mean(Ct) P = 0.0046
of metastases at presentation (RCC), time from lung
CD3E
Immune cell CD4
CD8A
metastasis diagnosis to surgery (RCC), disease-free inter-
populations CD68 val (RCC), alkaline phosphatase, hemoglobin, neutro-
Mean(Ct) P = 0.0094
phils, and platelets levels (RCC). The lower limit of
IFNG
TH1 IL12A normal was used for hemoglobin (cutoff values: men
IL12B
orientation 1L18
TBX21
13 g/dL and women 12 g/dL) and the upper limit
LTA
(ULN) was used for alkaline phosphatase (cutoff value:
Mean(Ct) P = 0.36
80 U/L), neutrophils (cutoff value: 7500/mm3), and
IL4
TH2 IL5
IL10
platelets (cutoff value: 400,000/mm3). With respect to
orientation IL13
immune cell densities and number of metastases, the
Mean(Ct) P = 0.16
"minimum P value" approach was used to determine the
C3
FN1 cutoff for the best separation of patients referring to their
IL3
IL6
IL7 * OS outcome (outcome-oriented approach). Because the
TNF
CSF1 P values obtained might be overestimated, OS log-rank
IL1A
Inflammation IL8 P values were corrected using the formula proposed by
IL1B
IL17
PTGS2 Altman and colleagues (25) and using 10-fold cross-
SELE
CSF3 validations as recommended by Faraggi and colleagues
STAT3
* (26). The confidence interval was important around
Mean(Ct) P = 0.130
ACE
the optimal P value (Supplementary Table S3). We have
Angiogenesis CD34
VEGF * also ensured that the significance established at the
Mean(Ct) P = 0.165 optimal cutoff remained valid at the quartiles (data-
Immuno- TGFB1
CTLA4
oriented approach). A P value less than 0.05 was consid-
suppression IL10
ered statistically significant. Independent parameters
Mean(Ct) P = 0.126
identified at univariate analysis as possibly influencing
GLNY
GZMB outcome (P < 0.1) were introduced in a multivariate Cox-
Cytotoxicity PRF1
IL15 proportional hazards regression model. All analyses were
Mean(Ct) P = 0.018 conducted with Prism 5 (GraphPad), Statview (Abacus
CCL2
CCL3 Systems), and the R (http://www.r-project.org/).
CCL5
Chemokines/ CCR2
CCR5
chemokine
CCL19
Results
receptors CCR4
CCR7
CXCL10
The densities of immune cells correlate with OS in lung
CXCL11
CXCR3
metastases from colorectal carcinoma
High CD8+/ Low CD8+/
Because densities of CD8 T cells and DC-LAMP mature
DC-LAMP+ DC-LAMP+ dendritic cells in primary tumors correlate with survival (1),
densities densities
we counted these cells in lung metastases from 140 colo-
rectal carcinoma patients. We also quantified NKp46 NK
Figure 3. (Continued ) E, expression of genes related to immune cell cells as a marker of innate immune response. High densities
populations, TH1/TH2 orientations, inammation, angiogenesis, of infiltrating CD8 T cells (Fig. 1A) and mature dendritic
immuno-suppression, cytotoxicity, chemokines/chemokine receptors cells (Fig. 1B) were associated with prolonged OS (P 0.039
according to the CD8 and DC-LAMP cell densities (high/high vs. and 0.001, respectively). Combination of these 2 immune
low/low) in lung metastases from RCC. Expression levels of genes were
determined using threshold cycle (Ct) values normalized to actin B
parameters allowed to identify patients with better outcome
[ACTB] (DCt). We used MannWhitney test to identify genes with (CD8high/DC-LAMPhigh; Fig. 1C, P 0.008). NKp46 cell
signicantly different levels of expression among patient groups (high vs. density did not predict clinical outcome (P 0.12; Fig. 1d).
low).  , P < 0.05 for individual gene expression. Significance was established at the optimal cutoff, but
remained valid at quartiles including the median (Supple-
or similar methods. Correlations were evaluated by the mentary Table S3). The quantification of CD8 T cells
Spearman test. Overall survival (OS) curves were estimat- separately in the center of the tumor and the invasive margin
ed by KaplanMeier method and differences between the regions yielded similar results (Supplementary Fig. S1).
groups of patients were calculated using the log-rank test. Univariate analysis of other clinical and pathological para-
The start of follow-up for OS was the time of lung surgery. meters is reported in Table 1. At multivariate analysis (Table
In addition to mature dendritic cells, CD8 T cells, and 1), immune pattern (CD8/DC-LAMP densities) of metas-
NK cells densities, the following available clinical para- tases was the strongest independent predictor of survival.
meters were tested: initial stage (colorectal carcinoma), As reported in colorectal carcinoma primary tumors (3),
completeness of resection at pulmonary level, number of gene expression analyses revealed that a strong CD8 and
lung metastases, presence of extrathoracic metastases at DC-LAMP 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 asso-
ciated with TH2 orientation, inflammation, angiogenesis,
or immunosuppression were not correlated with the CD8/
DC-LAMP densities. However, individual gene expression
of VEGF was inversely correlated with CD8/DC-LAMP
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 14) oper-
ated 1 to 9 months after the initial metastatic surgery, and/or
relapsing metastasis occurring 14 to 52 months after sur-
gical removal of the lung metastasis (n 12; Fig. 2A).
Densities of CD8 (Fig. 2B), DC-LAMP (Fig. 2C), and
NKp46 (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. 2BD).
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 25; Fig.
2E). Primary colorectal carcinoma differed from lung
metastases by significantly higher density of CD8 T cells
(P < 0.05; Fig. 2F), but the density of each cell type was
positively correlated between the primary and the metastat-
ic tumors (Fig. 2FH for CD8, DC-LAMP, and NKp46 (Fig. 5A). TLS contained a B-cell follicle, a T-cell zone, and
cells, respectively). We had access to a small number (n 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 metas-
tases. Patients with high densities of infiltrating CD8 T cells
(Fig. 3A) or DC-LAMP cells (Fig. 3B) have reduced survival
(P 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 NKp46 cells was associated with improved survival (P
0.002; Fig. 3D). Separate analysis of the CD8 and NKp46
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). Uni-
variate proportional hazard Cox analyses revealed that the
immune pattern (CD8/DC-LAMP densities), NKp46 cell
density, presence of metastases at presentation, and disease-
free interval were the only prognostic factors of patients
4086 Clin Cancer Res; 19(15) August 1, 2013
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survival in our cohort (Table 1). Our data also suggest that
hemoglobin and thoracic lymph node invasion tended to be
associated with survival (P 0.061 and 0.086, respectively).
In the resulting multivariate proportional hazard Cox mod-
el, DFI and immune pattern were independent prognostic
factors (P 0.0067 and 0.0039, respectively; Table 1).
A strong CD8/DC-LAMP 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 expres-
sion was positively correlated with CD8/DC-LAMP infil-
tration, 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-LAMP,
CD8, and NKp46 cells in the primary tumor and in the
corresponding lung metastasis (n 24; Fig. 4AD), indi-
cating 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 CD8, DC-LAMP, and NKp46 cell densities in
lung metastases have different clinical impacts in colorectal
carcinoma and RCC, we compared their microenviron-
ments. Histologic analyses revealed profound differences
between colorectal carcinoma and RCC lung metastases. We
found glands, often necrotic, in an abundant and collage-
nous stroma surrounded by a high density of tertiary lym-
phoid 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
PNAd high-endothelial venules (Fig. 5B).
We found similar densities of CD3 and CD8 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 carci-
noma 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 CD8,
DC-LAMP, and NKp46 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
Clinical Cancer Research
 Published OnlineFirst June 19, 2013; DOI: 10.1158/1078-0432.CCR-12-3847

In Situ Immune Reaction in Lung Metastases

A Primary tumor versus lung metastasis:

Figure 4. CD8 T cells, DC-LAMP
Lung
mature dendritic cells, and NKp46 Primary
metastasis
tumor
NK cell densities in metastases and
in primary RCC tumors. A, surgical
treatment for primary RCC and
their lung metastases. BD, RCC
primary tumors were less inltrated
by DC-LAMP cells than lung
metastases. R values show the
positive correlations (0.5 < R < 0.9
B R = 0.689 C R = 0.547 D R = 0.817
ns * ns
and P < 0.05, Spearman test) 10,000 15 800
between primary tumors and

Number of DC-LAMP+ cells/mm2

Number of NKp46+ cells/mm2

lung metastases according to the
Number of CD8+ cells/mm2

CD8, DC-LAMP, and NKp46 8,000

600
cell densities. PT, primary tumor;
10
LM, lung metastasis; ns, not 6,000
signicant;  , P < 0.05 (Wilcoxon
matched pairs test). 400
4,000
5
200
2,000

0 0 0
PT
L M PT
LM PT -L
M
C- C- C- C- C- C
RC RC RC RC RC 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 metas-
tases. 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 CD8 T cells) in metastases from colo-
rectal carcinoma and RCC, although with opposite impact
on OS. In our cohorts of oligometastatic surgically treated
patients, the strongest prognosticator was the immune
pattern, that is CD8 and DC-LAMP cell density combi-
nation, as reported for many primary tumors (113, 23,
27, 2931). 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 incom-
plete data collection (especially for laboratory values which
were difficult to collect in a retrospective analysis), conclu-
sions 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 CD8 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 0.15 and 0.01 for CD8
and DC-LAMP cells, respectively; data not shown) than in
lung metastases from colorectal carcinoma (P 0.039 and
0.001 for CD8 and DC-LAMP cells, respectively; data not
shown).
Clinical significance of CD8 T cells density seems to be
contrasted, according to primary tumors origin. Although
we found similar densities of CD8 T cells in both colorectal
carcinoma and RCC metastases, the prognostic value of

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Remark et al.

these cells was different. Similar conflicting observations

A Colorectal cancer -
Lung metastasis
B Colorectal cancer -
Lung metastasis
Renal cell carcinoma -
Lung metastasis
Renal cell carcinoma -
Lung metastasis
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 nonsmall
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 expres-
sion 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 colo-
rectal carcinoma and RCC, which share a TH1 profile, but
the latter exhibit also a TH2, inflammatory, and immuno-
suppressive 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 (3739), which could initiate an impaired
T-cell response in RCC, resulting in poor prognosis. Inter-
estingly, VEGF gene expression was positively correlated
with high CD8 and DC-LAMP infiltration in RCC lung
metastases and with low CD8 and DC-LAMP infiltra-
tion in colorectal carcinoma lung metastases. Because it
has been suggested that VEGF may induce non-coordi-
nated immune responses (27), affect cytotoxic TH1 adap-
tive immune responses (39, 40) and contribute to the
progression of malignant disease, the correlation between
CD8/DC-LAMP 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/DC-
LAMPhigh 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 magnication: 40

Figure 5. Comparison of the immune contextures in colorectal carcinoma
and RCC lung metastases. A, representative pictures of colorectal
carcinoma and RCC lung metastases [hematoxylineosinsafran (HES)
and 200. TLS, tertiary lymphoid structure; T, tumor; S, stroma. B,
location and organization of CD20 B-cell follicles (red) surrounded by
high-endothelial venules (blue), DC-LAMP expressing mature dendritic
cells (red, black arrows), CD3 T cells (red), CD8 T cells (red), and
NKp46 NK cells (red) in colorectal carcinoma (left) and RCC (right) lung
metastases. Original magnication: 200 and 400.

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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 CD3+ T cells: CD8+ T cells:

D
2.0 1:1 2.0
ns ns High Low
10,000 10,000 expression expression
CD68
Immune cell *
CD3E
Number of CD3+ cells/mm2

Number of CD8+ cells/mm2

populations CD4 ns
CD8A

1,000
IL18
TBX21
Th1 IFNG
1,000 orientation IL12A ns
IL12B
LTA

100 IL5
Th2 IL4 *
orientation IL10 ns
IL13

FN1
C3
VEGF
100 10 STAT3
CSF1
LM LM LM LM ACE
*
C- C- C- C- CD34
CR RC CR RC
IL6
Inflammation IL7
TNF
and IL3
angiogenesis IL8

DC-LAMP+ mature NKp46+ NK cells:

IL1A *
IL1B
PTGS2
***
*** SELE ns
1,000 CSF3
10 IL17
Number of DC-LAMP+ cells/mm2

TGFB1
Number of NKp46+ cells/mm2

Immuno- CSF1 *
suppression CTLA4
ns
IL10

1 100
GZMB
GLNY
Cytotoxicity PRF1
IL15
*
CCR2
CCL2
0.1 10 CCR5
*
CCL3
CCL5
Chemokines/
CCR4
chemokines
receptors
CCL19
CXCL11 *
CXCR3
0.01 1 CXCL10
CCR7
ns
LM LM M M
C- C- C-
L
C-
L
Colorectal Renal cell
CR RC CR RC carcinoma - LM carcinoma - LM

Figure 5. (Continued ) C, quantication of CD3 , CD8 , DC-LAMP , and NKp46 cells in lung metastases from colorectal (colorectal carcinoma-LM,
n 140) and renal cell carcinoma (RCC-LM, n 52). Whiskers length represents 10 to 90 percentile. ns, not signicant;    , P < 0.0001 (MannWhitney 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 signicant;  , P < 0.05 (MannWhitney test).

acute/chronic inflammatory microenvironment, are often We found that colorectal carcinoma and RCC have a
reported to have some efficacy in metastatic renal cell correlated pattern of DC-LAMP, CD8, and NKp46 cells,
carcinoma (43). from primary tumor to relapsing metastasis, which could
The Von Hippel Lindau phenotype, often found in RCC, reflect, either a potential "imprinting" of the immune
may also be involved in the shaping of peculiar tumor microenvironment by the tumor cells or the possibility that
microenvironments, through induction of hypoxia, pro- the immune contexture in the primary tumor, results in
duction of VEGF, induction of regulatory immune circuits "educated" immune cells that are recalled in the metastatic
(4447), and increased sensitivity of tumor cells to NK-cell sites.
lysis (48). It may also influence differently the stroma In conclusion, our findings highlight the fact that during all
characteristics, the vascularization, or the collagen content steps of cancer development, reciprocal interactions occur
which could also impact on the migration, organization, between immune and cancer cell and are critical for patients
and functionality of intratumor immune cells (49). Togeth- survival. The immune signature seems to be a phenotypic
er, these data may explain the negative clinical impact of the marker for the disease and is remarkably reproduced between
adaptive immune pattern at the primary and advanced primary and metastatic sites in the same patient. The immune
stages of RCC. contexture affects OS in lung metastases from colorectal

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 Published OnlineFirst June 19, 2013; DOI: 10.1158/1078-0432.CCR-12-3847
Remark et al.
carcinoma and RCC, and the analysis of the immune pattern
might be useful to guide therapeutics (50).
Disclosure of Potential Conicts 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. R
egnard, 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. Fl
ejou, L. Gibault, J.-F. R
egnard, O.N. Pages, S.
Oudard, D. Damotte
Analysis and interpretation of data (e.g., statistical analysis, biosta-
tistics, 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-Frid-
man, W.H. Fridman, D. Damotte
Administrative, technical, or material support (i.e., reporting or orga-
nizing data, constructing databases): R. Remark, L. Crozet, A. Cazes,
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Grant Support
This work was supported by Institut National de la Sant
e et de la
Recherche M
edicale (INSERM), Universit
e Paris-Descartes, Universit
e Pierre
et Marie Curie, Institut National du Cancer, Canc
eropole Ile de France, and
Labex Immuno-oncology (2011-1-PLBIO-06-INSERM 6-1, PLBIO09-088-
IDF-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
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Clin Cancer Res; 19(15) August 1, 2013 4091
 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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