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From the immune contexture to the Immunoscore: the role of


prognostic and predictive immune markers in cancer
Helen Angell1,2,3 and Jerome Galon1,2,3
The inherent complexity of multifactorial diseases such as
cancer renders the process of patient prognosis and prediction
of response to therapy extremely difficult. Many markers,
signatures, and methods have been described to evaluate the
prognosis of cancer patients, yet very few translate into the
clinic. Systems biology approaches have facilitated analysis of
the complex interaction between tumors and the host-immune
response, and allowed the definition of the immune contexture.
Here we review the potential of the immune contexture,
quantified by the Immunoscore, to provide a statistically strong
parameter for prognosis. Finally we introduce the concept that
the host-immune reaction could be the critical element in
determining response to therapy. The effect on the immune
response could be the underlying factor behind many of the
predictive makers.
Addresses
1
INSERM, U872, Laboratory of Integrative Cancer Immunology, Paris,
France
2
Universite Paris Descartes, Paris, France
3
Centre de Recherche des Cordeliers, Universite Pierre et Marie Curie
Paris 6, Paris, France
Corresponding author: Galon, Jerome (jerome.galon@crc.jussieu.fr)

Current Opinion in Immunology 2013, 25:xxyy


This review comes from a themed issue on Cancer immunotherapy:
clinical translation
Edited by Tom Gajewski and Ton Schumacher

0952-7915/$ see front matter, Published by Elsevier Ltd.


http://dx.doi.org/10.1016/j.coi.2013.03.004

Introduction
The evolution of cancer is greatly influenced by the
complex milieu and microenvironment in which it develops, harboring tumorcell interactions with host endothelial cells, fibroblasts, blood vessels, lymph vessels,
immune cells, cytokines, chemokines and products of
cellular metabolism. The inherent complexity of such a
multifactorial disease renders the process of patient prognosis and prediction of response to therapy extremely
difficult. A good prognostic marker is a biomarker that
provides information on the likely course of the disease in
an untreated individual or regardless of treatment. However, a predictive biomarker can be used to identify
subpopulations of patients who are most likely to respond
to therapy.
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Definition of immune contexture


Histological analysis of human tumors, in particular colorectal tumors (CRC), has highlighted the importance of
the combination of immune variables. Tumor immune
infiltrates include macrophages, dendritic cells (DC),
mast cells, natural killer (NK) cells, nave and memory
lymphocytes, B cells and effector T cells. Analysis of
these in situ immune components and their organization
has revealed large heterogeneity between tumor types
and also a broad patient-to-patient diversity. The role of
the immune system in controlling tumor progression and
its effect on tumor escape, and thus patient prognosis, is
becoming increasingly understood. We have previously
described these major immune parameters, associated
with survival, as the immune contexture [1]. The
immune contexture is defined as the type, functional
orientation, density and location of adaptive immune
cells within distinct tumor regions [1,2,3,4]. As summarized in Figure 1a, the parameters that establish the
immune contexture comprise the density of CD8+ cytotoxic T lymphocytes (CTL) and memory T cells
(CD45RO+), their location at the tumor center (CT)
and invasive margin (IM), combined with the quality
of tertiary lymphoid structures (TLS) and additional
functionality entities such as TH1-related factors (IFNG,
Tbet, IRF1, IL12), chemokines (CX3CL1, CXCL9,
CXCL10, CCL5, CCL2), adhesion molecules (MADCAM1, ICAM1, VCAM1) and cytotoxic factors (granzymes, perforin, granulysin).

Immune markers as prognostic markers


Traditionally, the anatomic extent of the tumor burden in
CRC and all solid tumors has been the most important
prognostic factor (Figure 1b). Data on the tumor burden
(T), combined with the presence of cancer cells in draining and regional lymph nodes (N) and evidence of
metastases (M), amalgamate to provide tumor staging
(AJCC/UICC-TNM classification). TNM stages estimate
patient postoperative outcome and the rationale for adjuvant therapy. Despite the prognostic power of this staging
system, it is becoming recognized within the cancer
community that clinical outcome can significantly vary
among patients within the same stage. The current classification provides limited prognostic information, and does
not predict response to therapy. Advances in this field
have alluded to the importance of the immune prevalence
within the tumor microenvironment. A strong lymphocyte infiltration has been reported to be associated with an
antitumor response and improved clinical outcome. This
correlation between the prevalence of tumor infiltrating
Current Opinion in Immunology 2013, 25:17

Please cite this article in press as: Angell H, Galon J. From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer, Curr Opin Immunol
(2013), http://dx.doi.org/10.1016/j.coi.2013.03.004

COIMMU-1196; NO. OF PAGES 7

2 Cancer immunotherapy: clinical translation

Figure 1

(a)

(b)

CD8+

IV

N+

M+

Immunoscore:
I0

I1, I2, I3

I4

T1

Cytokines

Chemokines

III

Cytotoxic
factors

Th1

CD45
RO+

(c)

Tumour Stage:
I
II

T2

Adhesion
molecules
TLS

Tumour centre

T3

Invasive margin
T4

Tumour
OS
DFS
Immune
Contexture
Type
Location
Density
Functional
orientation

OS
DFS

OS
DFS
Key Parameters
CTLs (CD3+CD8+)
Memory T cells
(CD3+CD45RO+)
Tumour centre (CT)
Invasive margin (IM)
Presence and quality of TLS
Continuous
Th1 cell-associated factors
Cytotoxic factors
Chemokines, cytokines
Adhesion molecules

Characteristic : complex

Immune Reaction

OS
DFS

TNM
Staging

Key Parameters

Immunoscore

Key Parameters

Tumour (T)

Longitudinal extent of tumour burden

Type

CTLs (CD3+CD8+)
Memory T cells (CD45RO+)

Lymph node
(N)

Presence of cancer cells in draining


and regional lymph nodes

Location

Tumour centre (CT)


Invasive margin (IM)

Metastases
(M)

Evidence of distant site metastases

Density

Predefined cut points

Cox analysis
(ref. 23)

DFS
HR/P-value

Cox analysis
(ref. 23)

DFS
HR/P-value

OS
HR/P-value

DSS
HR/P-value

1.38/0.09 ns 1.18/0.29 ns 1.43/0.10 ns


Characteristic : current standard classification

OS
HR/P-value

DSS
HR/P-value

0.64/<0.0001 0.71/<0.0001 0.63/<0.0001


Characteristic : powerful, simple
Current Opinion in Immunology

Schematic representation and accompanying key parameters distinguishing the (a) immune contexture, including the tumor center, invasive margin
and tertiary lymphoid structures (TLS); (b) the TNM classification system, highlighting the current staging strategy and (c) the Immunoscore, illustrating
the importance of the immune reaction regardless of tumor burden. CT, tumor center; CTL, cytotoxic T lymphocyte; DSF, disease free survival; DSS,
disease specific survival; IM, invasive margin; OS, overall survival.

immune cells and patient outcome has been well documented in melanoma [58], ovarian [9,10,11], head and
neck [1214], bladder [15,16], breast [1720], urothelial
[16], colorectal [2,21,22,23,2440], renal [41], prostatic
[4244] and lung cancer [4549]. The majority of studies
have demonstrated that high densities of CD3+ T cells,
CD8+ cytotoxic T cells and CD45RO+ memory T cells
are associated with a longer disease free survival (DFS)
and/or improved overall survival (OS) [3].

Immunoscore as prognostic marker


Accumulating data, collected from large cohorts of
human cancers, have demonstrated the impact of
immune-classification, which has a prognostic value
that may add to the significance of the AJCC/UICC
TNM-classification. Derived from the immune contexture, a simple and powerful immune-classification has
been termed the Immunoscore (Figure 1c). The
Immunoscore (I) is based on the numeration of two
lymphocyte populations (CD3/CD45RO, or CD3/CD8
or CD8/CD45RO) quantified within the CT and IM.
These parameters provide a scoring system ranging
Current Opinion in Immunology 2013, 25:17

from Immunoscore 0 (I0), which has low densities of


both cell types in both regions; to Immunoscore 4 (I4),
having high densities of both cell populations in both
regions. The prognostic value of using these immune
criteria was demonstrated in patients with early stage
CRC (AJCC/UICC TNM stage III CRC) to predict
survival and relapse [27]. The five Immunoscore groups
were associated with dramatic differences in DFS and
OS (P < 0.0001). Five years after diagnosis, only 4.8%
of patients with high densities of CD8 and CD45RO
cells had tumor recurrence, and 86.2% survived. In
contrast, the tumor recurred in 75% of patients with
low densities of these cell populations and only 27.5%
survived [27].
Combined evidence illustrates the dependency of the
current staging stratification on factors of the immune
response. In particular, the nature, functional orientation,
density, and location of adaptive immune cells within
distinct tumor regions have been illustrated to have a
prognostic value that may be superior to the TNMclassification. Cox multivariate analysis shows that tumor
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Please cite this article in press as: Angell H, Galon J. From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer, Curr Opin Immunol
(2013), http://dx.doi.org/10.1016/j.coi.2013.03.004

COIMMU-1196; NO. OF PAGES 7

Prognostic and predictive immune markers in cancer Angell and Galon 3

progression and invasion is statistically dependent on the


Immunoscore. Indeed, the immune pattern remained the
only significant criterion over the classical AJCC/UICC
TNM classification for DFS and OS [50]. In patients who
did not relapse, the density of CD8 infiltrates was inversely
correlated with T stage, whereas in patients with recurrence the number of CD8 cells was low, regardless of the T
stage of the tumor [23]. Thus, evidence supports the notion
to introduce immunological biomarkers, implemented as a
tool for the prediction of prognosis and response to therapy.
Incorporating the Immunoscore into traditional classification could result in a greatly improved prognostic and
potentially predictive tool [51,52]. Immunophenotyping
as part of routine diagnostic and prognostic assessment of
tumors may provide crucial novel prognostic information,
facilitate clinical decision-making including rational stratification of patient treatment and guide therapeutic strategies [53,54].

Additional prognostic markers


Prognostic markers are clinical measures used to estimate
an individual patients outcome, such as recurrence of
disease, and correlate with survival independently of
systemic therapy. These prognostic factors range from
simple measures such as the stage of disease or tumor
burden, to more complex markers, including genetic
mutations. Many of the important prognostic markers
have been established for numerous diseases, however,
the translation of new markers into the clinic is becoming
increasingly difficult. Established markers include the
amplification of the MYCN proto-oncogene as an
indicator of poor outcome in neuroblastoma [55]. The
number of lymph nodes has a strong influence on the
prognosis of recurrence-free survival in breast cancer
[56,57]. In addition, the mutational status of K-ras is also
used as a prognostic marker, for example in non small cell
lung cancer (NSCLC) [58].
The development of high throughput cDNA microarray
and tumor array technologies has led to the investigation of global gene and protein expression profiles,
which has begun to revolutionize the search for predictive and prognostic markers. The detection of subtle
changes in the genetic composition of different tumor
stages offers the possibility of defining more precise
clinical outcomes. Example predictors include the 70gene signature [59] and intrinsic-subtype classifiers
[60] in breast cancer, among many others in additional
disease settings [6164]. Unfortunately accuracies of
these predictors are still lower than 80% and thus it
remains difficult to identify a highly precise prognostic
biomarker for use across multiple patient cohorts. Studies have also attempted to find gene predictor lists in
ovarian cancer. Nine gene signatures were reported
with almost no common genes between each signature
(similarly to other cancer types) and none of these have
been implemented into the clinic.
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Genomics-based technologies have resulted in significant


advances in cancer diagnostics and prognostics, wherein
genomic instability, caused during dysregulated proliferation, is becoming increasingly investigated for its role in
diagnostics. A mutation in the adenomatous polyposis coli
(APC) gene occurs in approximately 60% of CRC
patients, as well as many others and is being used as a
biomarker to determine the stage, disease recurrence and
to monitor disease progression or response to therapy in
esophageal cancer [65]. An increase in the level of hypermethylated APC in the blood is associated with poor
survival.
The success and global utilization of a prognostic marker
requires key features in its implementation, including
feasible in routine settings, simple, inexpensive, rapid,
robust, reproducible, quantitative, standardized, and
powerful. The Immunosore appears to fulfill these
requirements [52]. Many markers, signatures, and
methods have been described to evaluate the prognosis
of cancer patients. Yet very few such markers and laboratory assays translate into clinical practice or reach the
statistical power of the Immunoscore. It is acknowledged
that additional markers may be used to further refine the
prognostic value of the Immunoscore.

Predictive markers
Prognostic markers are useful to assess the risk of an
individual patient, however, they do not provide information on whether a therapeutic regime will be
beneficial. Thus, we require additional biomarkers to
aid in treatment selection. For example, HER-2 overexpression has been associated with improved response to
trastuzumab (monoclonal antibody against HER-2) in
breast cancer [66,67].
Numerous different therapeutic regimes are currently
used to treat cancer, including cytotoxic therapies such
as chemotherapy and radiotherapy. Antibody therapies
include cancer centric approaches (tyrosine kinase inhibitor), tumor microenvironment directed strategies (antiVEGF antibodies [68]) or immune focused intervention
such as anti-PD1 [69,70] and the anti-CTLA4 antibody
ipilimumab [71,72,73]. In addition, the implementation
of small molecule inhibitors is increasing, for example
BRAF inhibitors [74]. Immunotherapeutic approaches
include Toll-like receptor (TLR) agonists, DC based
vaccines, peptide vaccines and adoptive T cell therapy
(ACT) [75]. It is becoming apparent that the majority of
the aforementioned therapies have an impact on the
immune system [3] (Figure 2a).
The efficacy of an anticancer therapy is primarily evaluated by its ability to inhibit the proliferation of tumor
cells. Whether or not the tumor immune contexture
predicts therapeutic response is of paramount importance
in patient clinical management [3]. One of the hurdles
Current Opinion in Immunology 2013, 25:17

Please cite this article in press as: Angell H, Galon J. From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer, Curr Opin Immunol
(2013), http://dx.doi.org/10.1016/j.coi.2013.03.004

COIMMU-1196; NO. OF PAGES 7

4 Cancer immunotherapy: clinical translation

Figure 2

(a) Cancer Treatment

(b) Biomarkers

Impact on immune response

Prognostic

Predictive

Major

Nonimmune
markers

Strong

Moderate

Weak

Immune
markers

Null
ACT TLR
ag

Vac

Abs Chemo Radio Small


mol

Therapy
Current Opinion in Immunology

(a) Schematic illustrating the impact of cancer treatments on the host immune response. (b) Diagrammatic representation depicting the potential
difference in overlap between immune and non-immune prognostic and predictive markers. ACT, adoptive cell transfer; TLR, toll-like receptor; ag,
agonist; Vac, vaccines; Abs, antibodies; Chemo, chemotherapy; Radio, radiotherapy; Small mol, small molecules.

related to the development of novel cancer immunotherapies is the absence of immune response biomarkers to
indicate the efficacy and kinetics of the antitumor
response. Current studies begin to highlight the interaction between tumor cell death, triggered by chemotherand
how
this
initiates
an
apy/radiotherapy
immunoadjuvant pathway that may contribute to the
success of the treatment. The prevalence of immune
infiltrates appears to predict clinical response to some
immunotherapies, including response to cancer vaccines
[76]. Breast cancer patients with a loss-of-function
mutation in TLR4 relapsed earlier after receiving anthracycline-based chemotherapy [77]. The survival benefit of
5-fluorouracil-based chemotherapy in CRC patients was
greatly improved with the presence of tumor infiltrating
lymphocytes (TILs, P = 0.02). Patients with increased
levels of perforation of the serosa wall also had improved
survival (P = 0.16) [78]. Since the presence of TILs
reflects an adaptive immune response and perforation
is associated with inflammatory response, results suggest
a potential interaction between the immune response and
chemotherapy.
To demonstrate the predictive value of a biomarker, one
needs a randomized trial in which a control arm of
untreated patients should be evaluated; otherwise, a
so-called predictive marker is likely to be prognostic as
well.

An immune predictive marker is likely to be a


prognostic marker
A limitation in the accuracy of many predictive markers is
the assumption that tumor progression is largely a cellautonomous process with a cancer cell centric focus.
Current Opinion in Immunology 2013, 25:17

However, the presence of immune cells may reflect a


distinct underlying biology of the tumor, since gene
expression profiling and confirmatory assays have
revealed the presence of a broad signature of inflammation [79]. This signature includes evidence for innate
immune activation, chemokines for T cell recruitment,
immune effector molecules, and expression of immune
regulatory factors. Of the aforementioned therapeutic
strategies, the related predictive markers could in fact
be a reflection of a pre-existing immune contexture and of
the immune response to the therapy. Are predictive
markers biased depending on their influence on the
immune contexture? Does the patients original immune
status skew survival outcome, regardless of therapy?
Because of this, with a few exceptions, it is our opinion
that a predictive marker is likely to also be a prognostic
marker (Figure 2b). Since tumor molecular features and
immune reactions are inter-related, a comprehensive
assessment of these factors is critical [80]. Examining
the effects of tumorhost interactions on clinical outcome
and prognosis clearly represents an evolving interdisciplinary field. Pathological immunity evaluation may provide novel information on prognosis and help identify
patient cohorts more likely to benefit from immunotherapy.

Conclusions
Systems biology and large-scale analysis are powerful
approaches to uncover mechanisms associated with tumor
progression and tumor recurrence. Integrative analyses
evaluating the immune infiltrate in human cancers are of
major importance. We have previously described these
major immune parameters, associated with survival, as the
immune contexture. The immune contexture is defined
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Please cite this article in press as: Angell H, Galon J. From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer, Curr Opin Immunol
(2013), http://dx.doi.org/10.1016/j.coi.2013.03.004

COIMMU-1196; NO. OF PAGES 7

Prognostic and predictive immune markers in cancer Angell and Galon 5

as the type, functional orientation, density and location of


adaptive immune cells within distinct tumor regions.
Compared to the immune contexture, no tumor
parameter associated with survival has been reported to
achieve the same level of significance in CRC. For routine
testing, a simple method has been proposed and defined
as the Immunoscore. The outcome of a current worldwide
task force to validate the Immunoscore in a multicenter
trial may result in its implementation as a new component
for the classification of cancer, designated TNM-I
(TNM-Immune). Furthermore, the immune contexture
and the Immunoscore may allow the prediction of
response to many immune-related therapies. Finally, it
is expected that defects in the immune contexture will
provide new therapeutic strategies to treat cancer.

Acknowledgements
We acknowledge all the scientists who made contributions to the area of
research reviewed here that were not cited due to space constraints. We
thank the members of the Laboratory of Integrative Cancer Immunology
for their invaluable contribution. The work performed in our laboratory was
supported by grants from the Institut National du Cancer (INCa), the
Canceropole Ile de France, INSERM, MedImmune, Qatar National
Research Fund under its National Priorities Research Program award
number NPRP09-1174-3-291, the European Commission (7FP, Geninca
Consortium, grant 202230), and the LabEx Immuno-oncology.

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Please cite this article in press as: Angell H, Galon J. From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer, Curr Opin Immunol
(2013), http://dx.doi.org/10.1016/j.coi.2013.03.004

COIMMU-1196; NO. OF PAGES 7

6 Cancer immunotherapy: clinical translation

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Please cite this article in press as: Angell H, Galon J. From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer, Curr Opin Immunol
(2013), http://dx.doi.org/10.1016/j.coi.2013.03.004