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Critical Reviews in Oncology/Hematology 110 (2017) 106–116

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Critical Reviews in Oncology/Hematology


journal homepage: www.elsevier.com/locate/critrevonc

Oncology reviews

Tumor infiltrating lymphocytes in gastrointestinal tumors:


Controversies and future clinical implications
Cinzia Solinas a , Grazia Pusole b , Laura Demurtas b , Marco Puzzoni b , Roberta Mascia b ,
Gilberto Morgan c , Riccardo Giampieri d , Mario Scartozzi b,∗
a
Molecular Immunology Unit, Institut Jules Bordet and Université Libre de Bruxelles, Boulevard de Waterloo, 127 1000 Brussels, Belgium
b
Medical Oncology, University of Cagliari, Policlinico Universitario ss 554 bivio Sestu km 4.5, Monserrato, CA, Italy
c
Medical Oncology, Skåne University Hospital, Lund, Sweden
d
Medical Oncology, UNIVPM, Ancona, Italy

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
1.1. The elements of the tumor immune environment: from innate to adaptive immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
1.2. The assessment of the immune infiltrate in tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
1.3. TILs and benefit from immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
2. TILs in bowel tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
2.1. Colorectal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
2.2. Anal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
3. TILs in gastric cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
4. TILs in liver tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
4.1. Hepatocellular carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
4.2. Biliary tract cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
5. TILs in pancreatic cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
6. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

a r t i c l e i n f o a b s t r a c t

Article history: Chronic inflammation following infections, autoimmune diseases or exposure to environmental irri-
Received 1 August 2016 tants plays a crucial role in tumor development and influences the host immune response to neoplastic
Received in revised form 6 November 2016 cells. The presence of an anti-tumor immune infiltrate is often associated with better outcomes in
Accepted 23 November 2016
gastro-intestinal primary cancers, particularly in those with high microsatellite instability (MSI-H).
Immunotherapeutic drugs inhibiting the PD-1 and PD-L1 pathway showed promising results in the treat-
Keywords:
ment of these patients in the metastatic setting. The aim of this review is to resume the role tumor
Tumor infiltrating lymphocytes
infiltrating lymphocytes (TILs) play in gastrointestinal tumors, underlining their potential value as a
Gastrointestinal tumors
Microsatellite instability
prognostic and predictive biomarker. TILs assessment could identify subsets of patients with high extent
Prognosis of TILs and better prognosis, that could be spared from adjuvant systemic treatments. Immune infiltra-
Immunotherapy tion parameters might be additional predictors of a greater benefit from the immunotherapy with the
immune checkpoint blockade.
© 2016 Elsevier Ireland Ltd. All rights reserved.

∗ Corrosponding author at: Oncologia Medica AOU Cagliari – Policlinico Universitario di Monserrato, Ss 554 bivio Sestu km 4500, 09042, Monserrato, Cagliari, Italy.
E-mail addresses: czsolinas@gmail.com (C. Solinas), grazia.pusole@gmail.com (G. Pusole), lau.demi@tiscali.it (L. Demurtas), marcopuzzoni@gmail.com (M. Puzzoni),
roboa@hotmail.it (R. Mascia), Gilberto.Morgan@Skane.se (G. Morgan), riccardo.giampieri81@gmail.com (R. Giampieri), marioscartozzi@gmail.com (M. Scartozzi).

http://dx.doi.org/10.1016/j.critrevonc.2016.11.016
1040-8428/© 2016 Elsevier Ireland Ltd. All rights reserved.
C. Solinas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 106–116 107

1. Introduction Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells exert
a cytotoxic activity. Tumor associated macrophages (TAM) and
The interaction between neoplastic cells and the immune sys- tumor associated neutrophils (TAN) can play both anti-tumor or
tem starts during the early phases of tumor development, when pro-tumor roles depending on their polarization (M1 and N1 or
strongly immunogenic cells are recognized and eliminated by M2 and N2 respectively). Dendritic cells (DC) and B lymphocytes
the immune response. In the equilibrium and escape phases, the are antigen presenting cells (APC). B cells can also produce anti-
most aggressive and less immunogenic cells are able to survive bodies (Ab), secrete cytokines and behave as regulatory cells. T
and finally evade immunity, tumor becoming clinically manifested regulatory lymphocytes (Treg) secrete soluble factors (IL-10 and
(Dunn et al., 2002). The presence of an extensive immune infil- TGF-␤, i.e.) and express high levels of the inhibitory immune check-
trate has been associated with improved patients outcomes (better point molecule CTLA-4 playing a suppressive role and regulating
survival and responses to treatments) in a variety of solid tumors the activity of some other immune cells at the tumor site.
[ovarian, colorectal (CRC), non-small cell lung cancer (NSCLC),
melanoma, HER2-positive and triple-negative (TN) breast cancer
1.2. The assessment of the immune infiltrate in tumors
(BC), . . .] (Hwang et al., 2012; Galon et al., 2006; Zeng et al., 2016;
Thomas et al., 2013; Salgado et al., 2015). More, the recent introduc-
In order to investigate the clinical significance of the immune
tion of new immunotherapeutic agents, the immune checkpoint
infiltrate in tumors, TILs evaluation has been done with various
inhibitors, showed promising results in the treatment of several
methods: gene signatures (analyzing the mRNA expression of spe-
advanced neoplasms, like melanoma, kidney, NSCLC, bladder can-
cific cytokines and genes involved in the immune response, with
cer and head and neck squamous cell carcinomas, giving rise to
activity and functional information) and pathological assessment
durable responses also in heavily pre-treated patients (Reck et al.,
on hematoxylin-eosin (H&E), immunohistochemistry (IHC) and
2016; Ferris et al., 2016; Larkin et al., 2015; Motzer et al., 2015;
immunofluorescence (IF) stained slides. H&E colored tumor sec-
Borghaei et al., 2015; Rosenberg et al., 2016). In the adjuvant set-
tions are routinely used, and allow the evaluation of the extent of
ting, higher rates of recurrence-free survival (RFS), overall survival
mononuclear inflammatory cells infiltration, which has a prognos-
(OS) and distant metastasis-free survival were observed in stage
tic and predictive significance in TN and HER2-positive BC, i.e. The
III melanoma patients treated with the anti-CTLA-4 ipilimumab
use of specific antibodies at IHC and IF (i.e.: anti-CD3, −CD20, −CD4,
(Eggermont et al., 2016). Nevertheless the activity of these drugs
−CD8, markers for T, B, CD4+ and CD8+ T lymphocytes respectively)
is limited to a proportion of subjects and is associated with non-
accurately identifies the various subpopulations of TILs (Salgado
negligible side effects, leading to a strong need for the identification
et al., 2015). Microscopic analysis gives information on TILs spatial
of the best responders to these treatments.
distribution (at the invasive margin, in the stroma or within cancer
cell nests) and density that have been shown to be prognostic in
1.1. The elements of the tumor immune environment: from
CRC (Galon et al., 2006).
innate to adaptive immunity

The tumor immune environment can be oriented towards 1.3. TILs and benefit from immunotherapy
an anti-tumor or alternatively to an inflammatory pro-tumor
response, depending on the (dynamic) balance of the different TILs have also been studied as predictive markers of response to
subpopulations of its tumor infiltrating leukocytes and their func- the new immune checkpoint blockade (ICB) therapies. Metastatic
tional status. For example, tumor associated neutrophils (TAN) melanoma patients who benefit more from the anti-PD-1 pem-
and macrophages (TAM) can be polarized in different popula- brolizumab had high extent of proliferating CD8+ TILs at the tumor
tions playing opposite roles, anti- (N1 and M1) and pro-tumor (N2 invasive margin, PD-L1+, PD-1+ immune cells and a more clonal
and M2) respectively (Gregory and Houghton, 2011; Kim et al., TILs TCR repertoire (Tumeh et al., 2014). The link between tumor
2016). Myeloid derived suppressor cells (MDSC) are immature genomics and better responses to immunotherapy has also been
myeloid cells that dampen T cell activation in a not antigen (Ag) investigated in metastatic melanoma patients treated with the
specific way, through different mechanisms (release of inhibitory CTLA-4 blockade, where a high mutational load and the pres-
molecules and deprivation of amino acids involved in the prolif- ence of specific neo-Ags predicted major clinical benefit from
eration of lymphocytes) (Kumar et al., 2016). Dendritic cells (DC) the treatment (Snyder et al., 2014). In NSCLC objective responses
are professional Ag presenting cells (APC) that migrate in and (OR), durable clinical benefit, and progression-free survival (PFS)
out of tissues and lymph-nodes; some DC subpopulations can be improved during treatment with pembrolizumab. These responses
immune-suppressive, others are not (Volovitz et al., 2016). Natu- were seen in the presence of an inflamed microenvironment
ral killer (NK) cells target those cells that have lost the expression together with activated effector T cells, in the presence of clonal
of MHC proteins (the so called missing-self) and can be also acti- neo-Ag and a high nonsynonymous mutation burden (McGranahan
vated by stressed-induced ligands that are upregulated by tumor et al., 2016; Rizvi et al., 2015). Pembrolizumab administered in
cells (namely the stress-induced self). Additionally, NK cells can first line in advanced NSCLC patients with tumors expressing PD-
shape the tumor microenvironment by producing cytokines that L1 on at least 50% of neoplastic cells gave rise to longer PFS
are involved in the differentiation, activation and recruitment of and overall survival (OS) with respect to standard platinum-based
other immune cells (Vivier et al., 2012). In the context of adap- chemotherapy (Reck et al., 2016). In metastatic CRC patients the
tive immunity, T and B lymphocytes drive an Ag specific response, mismatch repair (MMR) deficiency status predicted benefit from
through T cell (TCR) and B cell receptors (BCR) respectively. T lym- the anti-PD-1 treatment. This condition, caused by the inactiva-
phocytes are differentiated in various subpopulations with distinct tion of MMR-associated genes following either epigenetic silencing
profiles: some subsets of CD4+ helper T cells can have anti- (Th1, or germline mutations (with an additional somatic inactivation of
Tfh, . . .), pro-tumor (i.e.: Th2) or regulatory functions (Treg); cyto- the second allele) generates a large number of somatic mutations
toxic CD8+ T lymphocytes (CTL) mostly kill tumor cells and release (thousands versus hundreds that are found in mismatch-proficient
cytokines (Chen et al., 2016). B lymphocytes can present Ags, pro- tumors). These mutations accumulate in regions of repetitive DNA,
duce antibodies and cytokines and also behave as regulatory cells leading to microsatellite instability (MSI). In patients treated with
(Breg), inhibiting the immune response (Siliņa et al., 2014; Gorosito pembrolizumab, IHC expression of CD8 and PD-L1 by TILs was not
Serrán et al., 2015) (Fig. 1). significantly associated with the outcome(s) (Le et al., 2015).
108 C. Solinas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 106–116

Fig. 1. Principal elements of the immunologic network at the tumor site.

The aim of this review is to resume the role TILs play in gastroin- IHC biomarkers distinguishing these different components of the
testinal tumors, underlining their potential value as a prognostic immune infiltrate are lacking. Some cells with regulatory functions
and predictive biomarker. are Foxp3 negative. It has also been demonstrated the presence
of a subset of CD25+ Foxp3+ regulatory cells that were CD8+ T
lymphocytes (Churlaud et al., 2015).
2. TILs in bowel tumors
A global assessment of the immune infiltrate (through the
immunoscore) evaluating the presence of different lymphocytes
2.1. Colorectal cancer
subpopulations [CD3+ (marker for all T lymphocytes)/CD45RO+
(mostly expressed by memory T cells), CD3 + /CD8+ or
CRC is the second most common cancer in women, the third in
CD8 + /CD45RO + ] and their location within the tumor (in the
men for incidence, and the fourth most common cause of death
center or at the invasive margin) has a prognostic significance
from cancer worldwide, with a 5 year disease-related survival rate
and has been proposed as a new parameter to evaluate before
of 60% (Cancer Res, 2015; WHO, 2016). Mortality rates remain high
treatment decisions (Galon et al., 2012). In CRC type, density and
despite the introduction of new targeted therapies in advanced
location of immune cells in primary tumors were good predictors
stages of disease (Cutsem et al., 2014). In recent years, attention
of longer disease-free survival (DFS), as shown in large cohorts of
has turned to the role played by the host immune response in pri-
CRC patients by the use of both gene expression profiling and IHC
mary CRC. The first to analyse TILs in primary CRC were Naito et al.,
stainings. Patients with an increased expression of genes for Th1
showing that the presence of CD8+ T lymphocytes located within
adaptive immunity had the best prognosis and higher immune
tumor cells nests was associated with better overall survival (OS).
cell densities for CD3+, CD8+, GZMB+, and CD45RO+ cells within
These CD8+ T lymphocytes were positive for granzyme B (GZMB)
each tumor region (either in the center or at the invasive margin)
cytoplasmic granules and expressed Ki-67; these are markers for an
predicted longer DFS. These findings were confirmed in all the
activated cytotoxic phenotype (CTL) and for a higher proliferative
stages of the disease, immune parameters being independent good
activity, respectively (Naito et al., 1998) (Table 1). TNF-␣ expression
prognostic factors (Galon et al., 2006) (Table 1). High immunoscore
evaluated in CRC TILs identified a population of tumor Ag spe-
in early-stage CRC patients treated with adjuvant surgery alone
cific CTL, and represented an indicative parameter of activated T
predicts the risk of recurrence, with only 4.8% of relapses and a
cell function. An increased concentration of TNF-␣ in CRC tissues
5 year survival rate of 86.2% within this group (Pagès et al., 2009).
was an independent prognostic factor for improved OS, signifying
Four molecular subtypes of CRC have been identified through the
that the functional status of TILs represents an added value to be
evaluation of gene expression profiles, mutation, copy number,
investigated (Reissfelder et al., 2015) (Table 1). Interestingly the
methylation, microRNA and proteomics. Microsatellite instability
presence of Foxp3+ TILs in CRC was associated with a good progno-
immune (CMS1), accounting for around 14% of cases, represents
sis, despite previous findings in other gastro-intestinal (GI) tumors
a subgroup of hypermutated, microsatellite unstable CRC with
(Huang et al., 2014; Ladoire et al., 2011) (Table 1). Another study,
strong immune activation. CMS1 tumors are frequently diagnosed
showing a positive correlation between Foxp3+ TILs and preoper-
in females with right-sided lesions and the CMS1 population usu-
ative serum albumin levels, confirmed that high levels of Foxp3+
ally has a poor OS rate after relapse (Guinney et al., 2015). MSI-H
expression represented an independent prognostic factor for better
tumors have higher immunoscores, cytokines and chemokines
OS in stage II and III CRC patients (Wang et al., 2015a) (Table 1).
expression levels, and are mostly infiltrated by cytotoxic, Th1, Th2
The link between Foxp3+ TILs and a better outcome, in con-
and Tfh cells vs MSS tumors. Immunoscore considered together
trast with what most frequently found in other solid tumors could
with MSI and TNM staging system was the sole parameter able to
be secondary to differences in study methodologies or else in the
predict better disease specific survival (DSS), DFS and OS. Patients
different biological properties of each cancer, harboring different
with a high immunoscore had higher frequency of PD-1+ cells
phenotypes, growing in different organs and dealing with different
located in the center and at the invasive margin of the tumor,
host factors (since every tumor has its own microenvironment).
confirmed by a higher expression of PDCD1 mRNA, whilst the
Even though Foxp3 is at the moment the most specific biomarker
highest levels of its ligand CD274 were found in tumors with a
for Treg, there are several subpopulations of immune cells having
high expression of genes associated with memory and cytotoxic
regulatory functions (i.e.: natural and induced Treg) and specific
C. Solinas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 106–116 109

Table 1
An overview of the studies evaluating TILs in colorectal cancer and anal cancer.

SETTING YEAR DESIGN TILs RESULTS OUTCOME REFERENCES

CRC 1998 Retrospective CD8 Inverse correlation between CD8+ T cells Better OS (Naito et al., 1998)
After surgery within cancer cell nests and stage at diagnosis.
CD8 + T cells within cancer cell nests had a
significant impact on
OS [HR = 0.52; P = 0.016].
CRC 2004 Retrospective CD8 CD8+ TILs prognostic [HR = 0.43 95% CI Better OS (Prall et al., 2004)
After surgery 0.22–0.85; P = 0.009].
CRC 2006 Retrospective CD3 High CD3+, CD8+, CD45RO+ TILs in primary Better DFS and OS (Galon et al., 2006)
After surgery CD8 CRC correlated with improved DFS [P < 0.0001,
CD45RO P < 0.001, P < 0.0001] and OS [P < 0.0001,
P < 0.0001, P < 0.0001].
CRC 2008 Retrospective TILs TILs associated with improved OS in patients Better OS (Morris et al., 2008b)
After surgery treated with 5-FU chemotherapy [HR = 0.52
95% CI 0.30-0.91; P = 0.02]. Patients with TILs
gained more benefit from chemotherapy
[cumulative survival HR = 0.22].
CRC 2009 Retrospective CD8 High immune score (CD8CT/IM CD45ROCT/IM ) Better DFS, DSS and OS (Pagès et al., 2009)
After surgery CD45RO correlated with better DFS [HR = 0.34 95% CI
0.24–0.48, P < 0.0001], DSS [HR = 0.35 95% CI
0.24–0.52, P < 0.0001] and OS [HR = 0.54 95% CI
0.43–0.69, P < 0.0001].
CRC 2014 Meta-analysis Foxp3 Improved 1, 3 and 5-year OS if high Foxp3+ Better OS (Huang et al., 2014)
After surgery TILs [OR = 1.93 95% CI 1.51–2.48, 1.56 95% CI
1.31–1.87 and 1.65 95% CI 1.40–1.95,
respectively; P < 0.001].
CRC 2015 Retrospective CD8 Foxp3+ TILs and TNF-␣ are good prognostic Better OS (Reissfelder et al.,
After surgery CD4 parameters in CRC [P = 0.008 and P = 0.004 for 2015)
Foxp3 OS respectively].
TNF- ␣
CRC 2015 Retrospective Foxp3 High Foxp3+ TILs independent predictors of Better OS (Wang et al., 2015a)
After surgery better OS [HR = 0.543 95% CI 0.38 to 0.78;
P = 0.001].
Rectal cancer 2014 Retrospective CD8 High intraepithelial CD8+/Foxp3+ ratio before Tumor (Shinto et al., 2014)
Neo-adjuvant Foxp3 CRT predicted tumor regression [P = 0.0023]. regression.Better RFS
High CD8+ stromal TILs after CRT associated
with better 5-year RFS [P = 0.0058].
Rectal cancer 2015 Retrospective CD45RO High density of CD45RO+ TILs associated with T and N down staging. (Wang et al., 2015b)
Neo-adjuvant improved tumor (T) and nodal (N) down Better 3-year DFS
staging following 30 Gy/10f nRT. Prolonged
3-years DFS [P = 0.026, OR = 0.436] in
CD45RO + hi cases.
Anal cancer 2006 Retrospective CD3 High CD3+, CD4+ and GZMB+ TILs associated Worse NED-survival (Grabenbauer et al.,
Neo-adjuvant CD4 with worse NED-survival [P = 0.0189, P = 0.0115 2006)
CD8 and P = 0.0079 respectively].
CD68
Foxp3
GZMB
Anal cancer 2015 Retrospective CD8 High intra-tumoral and peri-tumoral CD8+ TILs Better 5-year DFS and (Hu et al., 2015)
(SCC) associated with better 5-year DFS [HR = 0.337; OS
Neo-adjuvant P = 0.01 and HR = 0.261; P = 0.004 respectively];
high peri-tumoral CD8+ TILs associated with
better 5-year OS [HR = 0.21; P = 0.025].
Anal cancer 2016 Retrospective TILs High TILs correlated with better RFS in 2 Better RFS (Gilbert et al., 2016)
Neo-adjuvant independent cohorts (n = 141 and n = 122)
[P = 0.004 and P = 0.033 respectively]. In the
analysis of combined cohorts p16+ patients
with high TILs had better RFS [P = 0.006].

CRC: colorectal cancer, OS: overall survival; HR: hazard ratio; TILs: tumor infiltrating lymphocytes; DFS: disease free survival; DSS: disease specific survival; TNF-␣: tumor
necrosis factor alfa; CRT: chemo-radiotherapy; RFS: relapse-free survival; NED: no evidence of disease.

T cells, as well as with B cell markers (Mlecnik et al., 2016). The combination arms respectively (Overman et al., 2016). Neverthe-
clinical activity of pembrolizumab has been investigated in a phase less even within MSS tumors, an increased overall lymphocytic
II trial evaluating the treatment of metastatic MSI-H and MSS score has been linked with higher neo-Ag load and a better CRC-
CRC patients. Their results showed that MMR–deficient (MSI-H) specific survival (at both univariate and multivariate analyses).
are more responsive to PD-1 blockade than MMR–proficient Among MSS, higher neo-Ag load and a higher degree of TILs were
(MSS) tumors. This can be explained by the increased number observed in polymerase epsilon (POLE)-mutated cases, suggesting
of mutation-associated neo-Ags resulting from MMR deficiency that this genomic alteration, generating “ultra-mutated” tumors
driving an higher immune-infiltration (Le et al., 2015). In this group could be determinant for immune infiltration (Giannakis et al.,
of metastatic MSI-H patients the combination of the anti-PD-1 2016). Pathogenic POLE-proofreading domain mutations occur
nivolumab with the anti-CTLA-4 ipilimumab has been tested in in 1-10% of CRC and are more frequent in younger with a 7.6%
the CheckMate-142 trial. Partial responses (PR) were observed in prevalence in patients aged <40 years old. These POLE-mutated
25.5% and 33.3% of the patients from the anti-PD-1 alone and the cases are characterized by an increase in CD8+ TILs extent and
110 C. Solinas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 106–116

higher expression of PD-1, PD-L1 and CTLA-4, with a favorable GC with EBV infection or MSI-H have been shown to have high
outcome observed particularly in stage II disease (Glaire et al., TILs infiltration and typically have a better prognosis, demonstrat-
2016). ICB therapeutic approach is underway in this group of ing the fundamental role played by the host response in this disease
patients. (Chiaravalli et al., 2005; Kim et al., 2015; Kang et al., 2016; Giampieri
While MSI-H and POLE mutations in CRC have been associated et al., 2016) (Table 2). Low density of CD4+ T helper and of CD8+ TILs
with high mutational burden and high TILs, RAS mutant tumors in radically resected GC have been correlated with a positive lymph
usually have a poor immune infiltration and a lower expression of node status and a higher stage at diagnosis. Improved survival has
immune checkpoint molecules, indicating that these mutations are been observed in the presence of high density of CD4+, CD8+ and
probably less immunogenic (Lal et al., 2015). NK TILs. Independent predictors of a poor OS were high densities of
Conversion to an inflamed phenotype in MSS colon cancers can TAN and TAM (Liu et al., 2015; Shen et al., 2010) (Table 2). Foxp3+
be achieved through the inhibition of mitogen-activated protein lymphocytes infiltration correlated with PD-L1 expression and the
kinase (MAPK) pathway together with a PD-L1 blockade, as shown combined presence of these two markers was associated with a
in a phase Ib study testing the combination of cobimetinib and worse prognosis (Hou et al., 2014) (Table 2). Although the gen-
atezolizumab. Higher CD8+ and MHC-I expression by tumor cells eral trend of considering CD8+ TILs as favorable and Foxp3+ TILs
resulted from a serial biopsy cohort in patients receiving these two as an unfavorable marker in radically resected GC, many studies
drugs (Bendell et al., 2016). showed contrasting conclusions (Choi et al., 2016; Haas et al., 2009)
TILs have also been shown to have a predictive value in rec- (Table 2). The role of the chemokine receptors that are involved in
tal cancers receiving a neo-adjuvant treatment. High CD8+/Foxp3+ TILs recruitment has also been investigated: chemokine receptor 3
intraepithelial ratio before chemo-radiotherapy (CRT) predicted (CXCR3) expression has been associated with increased CD4+/CD8+
tumor regression. High CD8+ stromal density after the treat- infiltration and improved OS (Li et al., 2015), high levels of the
ment was associated with a favorable clinical 5-year recurrence chemokine receptor 7 (CCR7) correlated with a positive nodal sta-
free survival (RFS). After CRT, stromal CD8+ TILs density doubled, tus and high Foxp3+ infiltration and it was associated with poor
whereas stromal Foxp3+ counts remained stable, demonstrating OS (Zhou et al., 2013) (Table 2). Inconclusive results came also
that treatments may influence the composition of TILs subpopula- from studies evaluating the prognostic significance of the immune
tions (Shinto et al., 2014) (Table 1). checkpoint molecule PD-L1 expression in the post-operative set-
Further predictor of tumor down-staging, of a better response ting (Kim et al., 2014; Zhang et al., 2015). CTLA-4 was associated
after pre-operative RT and of a longer DFS (at the univariate and with better outcomes (Kim et al., 2014). In the neo-adjuvant set-
multivariate analyses) was a high density of CD45RO+ TILs, with ting, patients receiving a pre-operative treatment with FOLFOX6
several studies confirming these findings (Morris et al., 2008a; had less intra-tumoral Foxp3+ lymphocytes and more DC infil-
Morris et al., 2008b; Prall et al., 2004; Wang et al., 2015b) (Table 1). tration in the residual disease with respect to patients receiving
surgical treatment without neo-adjuvant chemotherapy (NACT).
In the NACT group, higher Foxp3+ TILs and DC in the residual dis-
2.2. Anal cancer
ease positively correlated with the depth of tumor invasion and
low Foxp3+ together with high DC emerged as independent prog-
Anal tumors, including squamous cell carcinomas (SCC, the most
nostic factors for better OS (Hu et al., 2014) (Table 2). High CD3+
common), adenocarcinomas, basal cells carcinomas, melanomas
TILs assessed in the primary tumor were positively correlated with
and GIST account for around 2–3% of GI cancers. Around 90%
MMR deficiency and represented an independent prognostic factor
of anal cancers are attributable to human papillomavirus (HPV)
for better PFS and OS in a cohort of metastatic patients treated with
infection and further risk factors for the development of this dis-
a first-line platinum-based chemotherapy. Given their high neo-
ease are linked to immune suppression and autoimmune disorders
antigenic potential, as well as their high TILs, this subset of patients
(NCCN, 2017). A study evaluating a variety of TILs subpopulations
might represent the best candidates to receive immunotherapy
(CD3+, CD4+, CD8+ and CD68 + ) in biopsies from 38 patients with
with the immune checkpoint blockade (Le et al., 2015; Giampieri
anal carcinoma showed that CD3+ and CD4+ TILs and the expres-
et al., 2016). Immunotherapy with anti-PD-1 alone or in combina-
sion of GZMB at the tumor site were negative prognostic factors
tion with the anti-CTLA-4 showed activity and lasting responses in
(Grabenbauer et al., 2006). Lately, high levels of peri- and intra-
metastatic GC patients with MSI-H tumors. A complete response
tumoral CD8+ TILs in 40 patients diagnosed with SCC showed a
(CR) was also achieved in one patient with a MSS/EBV+ tumor
favorable effect on OS (Hu et al., 2015) (Table 1). Foxp3+ TILs did
receiving the dual blockade (Janjigian et al., 2016).
not influence prognosis (Grabenbauer et al., 2006) (Table 1).
High extent of TILs assessed at H&E predicted a better RFS in
two cohorts of 141 and 131 patients treated with radical RT+/−
4. TILs in liver tumors
chemotherapy (CT) for non-metastatic SCC of the anus and the anal
canal, stratified for HPV status (Gilbert et al., 2016) (Table 1).
4.1. Hepatocellular carcinoma

3. TILs in gastric cancer Hepatocellular carcinoma (HCC) accounts for up to 90% of all pri-
mary liver tumors, representing the sixth most common cancer(s)
GC is the fifth most common cancer in incidence and is the third and the second most common cause of cancer death worldwide,
most common type for mortality worldwide (Cancer Res, 2015). with its incidence increasing (Cancer Res, 2015). Chronic inflamma-
Its etiology is strongly related to chronic infections of Helicobacter tion induced by multiple etiological factors, mostly viral infections
pylori (HP) and Epstein-Barr virus (EBV) and immune regulation (HCV and HBV), alcohol consumption and metabolic syndrome,
signaling plays a crucial role in its pathogenesis (Prall et al., 2004). plays an important role in the malignant transformation of the hep-
The new molecular classification proposed by The Cancer Genome atocytes during the development of HCC and a variety of immune
Atlas (TCGA) (Hadjipanayis, 2014), identified four GC subtypes: cells (NK, APC, T and B lymphocytes) and cytokines (TGF-␤, IL-2)
the EBV infected tumors (EBViGC) showing amplification of CD274 participate in this process (Farazi and DePinho, 2006).
(PD-L1) and of PDCD1LG2 (PD-L2) genes, microsatellite unstable At time of diagnosis, only one third of patients are eligible
tumors (MSIGC) with elevated mutation rates, genomically stable for potentially curative therapies including resection, transplan-
tumors (GSGC) and tumors with chromosomal instability (CINGC). tation or local ablation [with median OS (mOS) rates of around 60
C. Solinas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 106–116 111

Table 2
A summary of the studies evaluating TILs in gastric cancer.

SETTING YEAR DESIGN TILs RESULTS OUTCOME REFERENCES

After surgery 2005 Retrospective CD3 High CD3+ and CD8 + TILs correlated with Better OS (Chiaravalli et al., 2005)
CD8 improved OS [failure time RR = 2.03;
P = 0.01/failure time RR = 2.09; P = 0.026
respectively].
After surgery 2009 Retrospective Foxp3 High density of stromal Foxp3+ TILs correlated Better OS (Haas et al., 2009)
with improved OS [0.25 95%CI 0.08-0.78;
P = 0.016].
After surgery 2010 Retrospective CD8 Foxp3+/CD8+ ratio correlated with worse OS Worse OS (Shen et al., 2010)
Foxp3 [HR = 2.827 95% CI 1.06–7.51; P = 0.037].
After surgery 2013 Meta-analysis Foxp3 High Foxp3+ TILs correlated with worse OS and Worse OS (Huang et al., 2014)
higher risk of recurrence.
After surgery 2013 Observational CCR7 High CCR7 expression correlated with high Worse OS (Zhou et al., 2013)
Foxp3 Foxp3+ TILs [P < 0.001]. High CCR7 expression
and high Foxp3+ TILs positively correlated with
lymph node metastases and were independent
predictors for worse OS [HR = 2.896 95% CI
1.39–7.28; P = 0.023/HR = 1.906 95% CI
1.21–4.24; P = 0.035 respectively].
After surgery 2014 Observational Foxp3 Significant correlation between Foxp3+ TILs Worse OS (Hou et al., 2014)
and PD-L1 expression [P = 0.01]. Foxp3+ TILs
and PD-L1 expression correlated with lymph
node metastases [P = 0.02] and with advanced
clinico-pathological stage [P = 0.04]. High
density of Foxp3+ TILs correlated with worse
OS [P = 0.015]. Over-expression of PD-L1 in
tumor cells and TILs was closely associated
with a lower OS rate [P = 0.033 and P = 0.021
respectively].
After surgery 2015 Retrospective CD8 CD8+/Foxp3+ ratio correlated with improved CD8+/Foxp3+ ratio (Liu et al., 2015)
Foxp3 OS [HR = 0.109 95% CI 0.015–0.790; P = 0.028]. correlated with better
High density of Foxp3+ TILs correlated with OS.
poor OS [HR = 5.580 95% CI 1.35-23.07; High density of Foxp3+
P = 0.018]. correlated with worse
OS
After surgery 2015 Retrospective CXCR3 High CXCR3 expression correlated with greater Better OS (Li et al., 2015)
CD4 CD4+ and CD8+ TILs infiltration [P = 0.032 and
CD8 P = 0.001 respectively].
High CXCR3 expression and high CD8+ TILs
correlated with improved OS. Patients with
advanced GC with low CXCR3 expression had a
0.5-fold increased risk of death [HR = 0.464 95%
CI 0.284–0.757; P = 0.002].
After surgery 2016 Observational Foxp3 High frequency of Foxp3+ TILs correlated with Better OS and DFS (Choi et al., 2016)
MDSC increased OS [HR = 0.047 95% CI, 0.006–0.372; Worse OS
P = 0.004] and DFS [P = 0.039].
High frequency of MDSC among total
examined cells correlated with decreased OS
[HR = 8.60 95% CI, 1.240–59.678; P = 0.029].
Neo-adjuvant 2014 Retrospective Foxp3 NACT decreased intratumoral Foxp3+ TILs and IntratumoralFoxp3 + TILs (Hu et al., 2014)
DC increased DC density [P = 0.007; P = 0.002, density correlated with
respectively]. Intratumoral Foxp3+ density worse OS.
negatively correlated with OS [HR = 7.599 95% Stromal DC density
CI 2.155-26.802; P = 0.002]. Patients with less correlated better OS
DC infiltration had a 4.6-fold increased risk of
death [HR = 4.617 95% CI 1.695–12.578;
P = 0.003].
Metastatic 2016 Retrospective CD3 Defective MMR related to TILs positivity, both Better OS and PFS (Giampieri et al., 2016)
factors showing an independent prognostic
role. High density of CD3+ TILs related to
improved OS [HR = 0.39 95% CI 0.26–0.58; P
< 0.0001] and PFS [HR:0.40; 95% CI 0.26–0.60
P = 0.0001].

TILs: tumor infiltrating lymphocytes; OS: overall survival; RR: risk ratio; HR: hazard ratio; CCR7: C-C chemokine receptor type 7; PD-L1: programmed death ligand 1;
CXCR3: C-X-C motif chemokine receptor 3; MDSC: myeloid derived suppressor cells; NACT: neoadjuvant chemotherapy; DC: dendritic cells; MMR: mismatch repair; PFS:
progression-free survival.

months]. In advanced disease, chemoembolization and sorafenib 7% of tumors had high TILs infiltration with a predominant pres-
represent the only available options with mOS of 26 and 11 months ence of T lymphocytes (mostly CD8+, followed by CD4+). Patients
respectively (Llovet et al., 2015). whose tumors showed this marked infiltration had significantly
The presence of intra-tumoral TILs is associated with a better higher OS and lower 5 year recurrence rates with respect to the
RFS. TILs were located within the trabeculae of neoplastic cells and TILs negative/low group (Wada et al., 1998) (Table 3).
were mostly composed of T lymphocytes (Shirabe et al., 1995). In a Similar findings were confirmed for the presence of high intra-
retrospective study of 163 consecutive surgical cases of HCC, only tumoral densities of both T and B cells which were associated with
112 C. Solinas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 106–116

Table 3
An overview of the studies evaluating TILs in liver tumors [hepatocellular carcinoma (HCC) and biliary tract cancer (BTC)].

SETTING YEAR DESIGN TILs RESULTS OUTCOME REFERENCES

HCC 1998 Retrospective CD8 High CD8+ TILs associated with better 5-year Better 5-year OS rate (Wada et al., 1998)
After surgery OS rate and lower recurrence rates [P < 0.01 and
and P < 0.01 respectively]. lower recurrence rates
HCC 2005 Retrospective IL-2 High mRNA expression of IL-2 associated with Better OS (Ikeguchi and Hirooka,
After surgery (mRNA) high CD8+ TILs. Patients with IL-2+ tumors had 2005)
CD8 a significant longer OS.
HCC 2007 Retrospective CD3 Low intra-tumoral Foxp3+ TILs and a Better DFS and OS (Gao et al., 2007)
After surgery CD4 concomitant high level of Granzyme B+ TILs
CD8 were independent prognostic factors for an
Foxp3 improved DFS [P = 0.0018] and OS [P < 0.0001].
GZMB
HCC 2014 Observational Circulating Increased CD19 + CD24 + CD39+ circulating Not evaluated (Shao et al., 2014)
After surgery Breg Breg in HCC patients were associated with
later UICC stages, venous infiltration, and
HBsAg positive status at diagnosis.
HCC 2015 Retrospective CD8 High intra-tumoral densities of both T and B Better OS (Garnelo et al., 2015)
After surgery CD19 cells was associated with better OS [P < 0.0001,
IFN-␥ HR = 4.8].
CD27
CD40
HCC 2016 Observational CD4 High circulating Treg correlated with a poor Worse DFS and OS (Wang et al., 2016)
After surgery CD25 DFS and OS [P = 0.037 and P < 0.001
TGF-␤1 respectively]. Liver cancer cells induced Treg
production by secreting TGF-␤1.
BTC 2003 Retrospective CD8 High intratumoral CD8+ TILs correlated with a Better OS (Oshikiri et al., 2003)
After surgery longer OS [P = 0.0001].
BTC 2009 Retrospective CD4 Increased expression of PD-L1 inversely Not evaluated (Ye et al., 2009)
After surgery CD8 correlated with CD8+ TILs. Inverse correlation
PD-L1 between TILs CD8+ and tumor grade and stage
PD-1 of disease.
BTC 2013 Observational CD4 High intraepithelial CD4+, CD8+,high total Better OS (Goeppert et al., 2013)
After surgery CD8 CD20+ and Foxp3+ TILs associated with a
CD20 longer OS [P = 0.002, P = 0.015, P = 0.032 and
Perforin P = 0.018].
Foxp3+
BTC 2016 Retrospective Circulating Low NLR (<3 vs ≥3) was associated with better Better RFS and OS (Lin et al., 2016)
After surgery neutrophils RFS and OS [P = 0.015 and P = 0.009].
and lym- Percentages in PD-1+ CD4+ and PD-1+ CD8+
phocytes; were higher in high NLR group.
PD-1+
CD4+
PD-1+
CD8+

HCC: hepatocellular carcinoma; BTC: biliary tract cancer; TILs: tumor infiltrating lympocytes; OS: overall survival; GZMB: granzyme B; DFS: disease free survival; Breg:
regulatory B cells; HBsAg: hepatitis virus B antigen; HR: hazard ratio; Treg: regulatory T cells; TGF-␤: transforming growth factor beta; NLR: neutrophil-to-lymphocyte ratio;
RFS: relapse free survival.

better OS (at univariate and multivariate analyses) in a retrospec- tissue samples. Co-culture of Treg isolated from TILs with autol-
tive study of 112 HCC patients. In this study, TILs subpopulations ogous circulating lymphocytes showed Treg ability in inhibiting
evaluation was done by flow-cytometry (on fresh tissues), IHC activity, proliferation, perforin expression and IFN-␥ secretion by
and IF (on FFPE tissue blocks) and quantitative polymerase chain CD8+ T cells (Unitt et al., 2005). Low intra-tumoral Foxp3+ with a
reaction (qPCR) was used for the evaluation of mRNA expression concomitant high expression of GZMB+ by TILs represent indepen-
of CD19, CD8A and IFNG (markers for B, CD8+ lymphocytes and dent prognostic factors for an improved DFS and OS, confirming
interferon (IFN)-␥, respectively). B lymphocytes were topographi- that a balance towards activated CD8+ lymphocytes reflects an anti-
cally close to T cells, and their presence was positively associated tumor immune response (Gao et al., 2007) (Table 3). Other studies
with an increased expression of GZMB and IFN-␥. Furthermore, the suggest that a high extent of Treg in association with high levels
expression of co-stimulatory molecules (CD27, CD40), regulating of transforming growth factor beta (TGF-␤) correlated with a poor
functional interactions between B and T cells, were linked to better prognosis (Wang et al., 2016) (Table 3). A study of 75 HCC patients
OS (Garnelo et al., 2015) (Table 3). demonstrated that increased levels of CD19 + CD24 + CD39+ circu-
High CD8+ TILs density evaluated by IHC has been positively lating Breg in HCC patients were associated with later UICC stages
correlated with IL-2 expression within a cohort of 59 surgically and venous infiltration at diagnosis. The presence of B TILs, identi-
resected HCC patients. High mRNA expression of IL-2 was asso- fied by IHC on FFPE blocks tissue samples showed that they were
ciated with high CD8+ T cells infiltration and patients with IL-2 mostly localized at the tumor margin rather than in the center of
positive tumors (56%) had a significant longer 5-year OS rate. The the tumor area. B cells infiltration was positively associated with
expression of IL-2 gene emerged as a prognostic biomarker at both hepatitis B surface Ag positive status, higher tumor size, venous
univariate and multivariate analyses and was independent from infiltration and later UICC stages (Shao et al., 2014) (Table 3).
tumor stage (Ikeguchi and Hirooka, 2005) (Table 3). A variety of immunotherapeutic agents (including DC-based
Treg, identified by the expression of CD4, CD25 (the receptor for therapies and ICB) has been tested in advanced HCC. Disease control
IL-2), CD45RO, and of high levels of intra-cellular CTLA-4 by flow with a time to progression of 6.4 months was obtained in patients
cytometry represented around 9% of isolated TILs from HCC fresh treated with the anti-CTLA-4 tremelimumab (Sangro et al., 2013).
C. Solinas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 106–116 113

Table 4
A summary of studies evaluating TILs in pancreatic cancer.

SETTING YEAR DESIGN TILs RESULTS OUTCOME REFERENCES

After surgery 2004 Retrospective CD4 High CD4+/CD8+ TILs correlated with better OS Better OS (Fukunaga et al., 2004)
CD8 [P = 0.0098].
After surgery 2009 Retrospective Circulating High MDSC associated with worse OS Worse OS (Gabitass et al., 2011)
MDSC [P < 0.001, HR = 1.22 95% CI 1.06–1.4].
After surgery 2015 Retrospective Tertiary High TLOs associated with better DFS and OS Better DFS and OS (Hiraoka et al., 2015)
lymphoid [P = 0.003 and P = 0.002].
organs
(TLOs)
After surgery 2016 Observational IL-22 IL −22 producing TILs increased in tumor Worse OS (Niccolai et al., 2016)
tissues and associated with worse OS.

TILs: tumor infiltrating lymphocytes; OS: overall survival; MDSC: myeloid derived suppressor cells; HR: hazard ratio; TLOs: tertiary lymphoid organs; DFS: disease free
survival.

In the phase 1/2 CheckMate 040 trial of advanced HCC, nivolumab to the organ site of origin (Goeppert et al., 2013) (Table 3). A study of
was administered in patients not amenable to curable resection, 102 consecutive patients diagnosed with ICC evaluated the prog-
that were intolerant or not previously treated with sorafenib (for nostic role of the neutrophil-to-lymphocyte ratio (NLR) obtained
refusal). In the HBV infected cohort an effective anti-HBV therapy by dividing the absolute number of neutrophils by the absolute
was given. OR were durable and observed at all dose levels and in count of lymphocytes in peripheral blood. Low NLR was associated
all etiologic subtypes (uninfected: sorafenib naïve/intolerant; unin- with better outcomes (RFS and OS at the univariate and multivari-
fected: sorafenib progressor; HCV and HBV). In the dose expansion ate analyses) and with a higher CD3+ and CD8+ TILs infiltration
cohort OS rate was of 71% at 9 months and safety profile was similar at the tumor site. A higher frequency of CD4+ PD-1+ and of CD8+
to what observed in other tumor types. Of note, PD-L1 expression PD-1+ TILs was seen in patients with high NLR, suggesting that the
did not impact on response to PD-1 blockade (Melero et al., 2016). evaluation of NLR in the peripheral blood could reflect the activ-
ity (anti- vs pro-) of the immune infiltrate facing with the tumor
4.2. Biliary tract cancers (Lin et al., 2016) (Table 3). A higher expression of PD-L1 by tumor
cells analyzed on 31 surgically resected ICC tumors was associated
Biliary tract cancers (BTC), are the second most common type of with more advanced stages of disease and with a poorer tumor dif-
primary hepatic neoplasms after HCC. They represent a heteroge- ferentiation. A large number of PD-1+ T lymphocytes (both CD4+
neous group of tumors, distinguished into: intrahepatic (ICC) and and CD8+) were observed in the stroma or within cancer nests. A
extrahepatic cholangiocarcinoma (ECC) and gallbladder adenocar- lower rate of CD8+ TILs was associated with more advanced disease
cinoma (GBAC). The prognosis of these diseases is unfavorable and stages (III–IV) and with a high tumor grade. CD8+ TILs presence was
diagnosis is often delayed. The majority of patients that undergo inversely correlated with the overexpression of PD-L1 (Ye et al.,
surgery develop recurrence after resection; CT and RT offer limited 2009) (Table 3).
benefits and there is a strong need for new alternative treatments
(Yamashita et al., 2008; Cabrera, 2013). One of the most com- 5. TILs in pancreatic cancer
mon risk factors associated with BTC development is the presence
of a pre-existing primary sclerosing cholangitis, which leads to a Pancreatic cancer (PC) is the seventh leading cause of cancer
chronic inflammation state, with an upregulation of several medi- mortality worldwide, representing an aggressive disease with a
ators which are believed to be involved in carcinogenesis and tumor poor prognosis (Cancer Res, 2015). This disease is often diagnosed
proliferation (Ishii, 2013). However, as stated before, the immune at advanced stages, and surgery represents a suitable solution for a
environment can also inhibit tumor growth and/or invasion, in the minority of patients. The chemotherapeutic drugs and the biologic
presence of a particular milieu of cytokines (IFN-␥ i.e.) and in the agents targeting the EGFR pathway (cetuximab and erlotinib) used
presence of some subpopulations of TILs that can exert a more in combination with gemcitabine confer a marginal benefit in terms
prominent anti-tumor activity. The presence of CD8+ T lympho- of OS (Ng et al., 2002; Hoff et al., 2011). New treatment options
cytes is linked to an anti-tumor immune response (Oshikiri et al., are urgently needed and immunotherapy could represent a valid
2003) (Table 3). Nevertheless their function could be inhibited by opportunity in particular subgroups of PC patients. Four molecu-
the interaction between PD-1 (expressed on CD8+ T cells follow- lar subtypes of PC have been recently identified: squamous, ADEX,
ing activation) and its ligands (PD-L1 and PD-L2) expressed on the pancreatic progenitor and immunogenic (associated with muci-
surface of tumor and immune cells within the microenvironment. nous non-cystic adenocarcinomas and carcinomas arising from
In a retrospective study of 435 BTC cases where TILs have been intra-ductal papillary mucinous neoplasia). Immunogenic subtype
evaluated by IHC, the most common immune cells detected at the showed high expression of immune gene signatures related to
tumor site were CD8+, followed by CD4+; Foxp3+ TILs were 12%. B infiltrating B and T cells (both cytotoxic and regulatory), with
cells and NK infiltrating TILs were rarely found, whilst macrophages upregulation of CTLA-4 and PD-1 pathways. High expression of
were frequently close to the tumor epithelium. The amount of lym- macrophage and T cell co-inhibition signatures in a subgroup of PC
phocytes (CD8+, CD4+, B and NK) was inversely proportional to the patients was associated with a poorer cumulative OS (Bailey et al.,
stage of disease, with higher extent observed in dysplastic lesions 2016). These observations highlight the significant role played by
and a progressive reduction in their amount observed in more the immune system among the different molecular subtypes of PC.
advanced lesions towards metastases. Interestingly the trend was Infiltration by both CD8+ and CD4+ TILs represented a favorable
different for macrophages whose number increased in invasive and prognostic factor after surgery in terms of OS rate. This condition
secondary lesions. Higher extent of CD8+, CD4+, CD20+ and Foxp3+ was also associated with less advanced TNM stages and inferior
intraepithelial TILs correlated with a longer OS. TILs impact on the tumor depth at diagnosis (Fukunaga et al., 2004) (Table 4). Through
outcome has been demonstrated for GBAC and ECC, not for ICC IL-10 and TGF-␤ secretion and CTLA-4 expression, Treg regulate a
patients. These results suggest that BTC might have important dif- variety of immunosuppressive mechanisms favoring tumor pro-
ferences with regard to density and composition of TILs according gression. An increased frequency of Treg has been associated with
114 C. Solinas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 106–116

a poor prognosis, a higher risk of recurrence of disease and a worse between TILs and an active immune response also suggests that this
survival after PC resection. Treg lymphocytes that are specific for parameter may also have a predictive role during immunotherapy.
the ␣-enolase Ag (that can also be expressed by PC cells) inhibit This suggestion, mainly deriving from studies in melanoma patients
the recruitment and the activation of the Th1 and Th17 effector T seems applicable to GI cancer patients as well. However further
cells (Amedei et al., 2013). High circulating levels of immunosup- investigations are needed to clarify whether the sole assessment
pressive MDSC and CD4+ CD25+ CD127low Foxp3+ Treg were found of TILs or together with the immune gene signatures evaluation
to be elevated in PC cancer patients. MDSC positively correlated could be used as valid biomarker(s) for the selection of the best
with high Treg levels, representing an independent factor for poor candidates for immunotherapy.
OS (Gabitass et al., 2011). The expression of intra-tumoral IL-22,
whose major source were IFN-␥ producing and perforin-mediated Conflict of interest
cytotoxic T cells therefore named Th22 cells was associated with
more advanced stages at diagnosis and worse OS (Niccolai et al., The authors declare that they have no conflict of interest.
2016) (Table 4). Also the presence of organized intra-tumoral and
peri-tumoral tertiary lymphoid organs (TLOs), has been correlated
with improved DFS and OS (at both univariate and multivariate Funding
analyses) (Hiraoka et al., 2015) (Table 4). An induction of similar
aggregates has been observed after the administration of a vaccine This review article did not receive any specific grant from fund-
given with or without cyclophosphamide in the neo-adjuvant set- ing agencies in the public, commercial, or not-for-profit sectors.
ting. After 2 weeks of treatment, patients underwent surgery and
analysis of tumor tissue sections. These showed that the majority Acknowledgement
of tumors (85%) had tertiary lymphoid organs and that PD-L1 was
expressed by immune cells in aggregates which were surrounded No writing assistance was utilized in the production of the
by PD-1+ cells. Treg infiltration was also induced by the vaccine and manuscript.
the balance between effector T cells and Treg was increased after
the treatment (Lutz et al., 2014).
References

Amedei, A., Niccolai, E., Benagiano, M., Bella, C.D., Cianchi, F., Bechi, P., et al., 2013.
6. Conclusions and perspectives Ex vivo analysis of pancreatic cancer-infiltrating T lymphocytes reveals that
ENO-specific Tregs accumulate in tumor tissue and inhibit Th1/Th17 effector
cell functions. Cancer. Immunol. Immunother. 62, 1249–1260, http://dx.doi.
Chronic inflammation following infections (such as EBV, HCV org/10.1007/s00262-013-1429-3.
and HBV), autoimmune diseases (i.e. inflammatory bowel disease) Bailey, P., Chang, D.K., Nones, K., Johns, A.L., Patch, A.-M., Gingras, M.-C., et al.,
or prolonged exposure to environmental irritants (like smoke or 2016. Genomic analyses identify molecular subtypes of pancreatic cancer.
Nature 531, 47–52, http://dx.doi.org/10.1038/nature16965.
obesity) plays a crucial role in the process of GI cancer develop- Bendell, J.C., Kim, T.W., Goh, B.C., Wallin, J., Oh, D.-Y., Han, S.-W., et al., 2016.
ment. Tumor-associated inflammation enhances neo-angiogenesis, Clinical activity and safety of cobimetinib (cobi) and atezolizumab in colorectal
promotes tumor progression and metastatic spread, causes local cancer (CRC). J. Clin. Oncol. 34.
Borghaei, H., Paz-Ares, L., Horn, L., Spigel, D.R., Steins, M., Ready, N.E., et al., 2015.
immunosuppression and further augments genomic instability. Nivolumab versus Docetaxel in advanced nonsquamous non–small–cell lung
Despite its pro-tumorigenic effects, inflammation also influences cancer. N. Engl. J. Med. 373, 1627–1639, http://dx.doi.org/10.1056/
the host immune response to tumors and can be manipulated NEJMoa1507643.
Cabrera, R., 2013. Treatment outcomes and prognostic factors of intrahepatic
through cancer immunotherapy (Grivennikov et al., 2010). The cholangiocarcinoma. Oncol. Rep., http://dx.doi.org/10.3892/or.2013.2290.
presence of CTL, memory T and Th1 cells has been generally asso- Cancer Res, 2015. Cancer Statistics for the UK. Cancer Res, UK, http://www.
ciated with better outcomes. The role played by other subsets cancerresearchuk.org/health-professional/cancer-statistics (accessed May 21,
2016).
of T cells is controversial and it can be dependent on the differ-
Chen, X., Du, Y., Lin, X., Qian, Y., Zhou, T., Huang, Z., 2016. CD4 + CD25 + regulatory T
ent balance of cells from the immune environment. It might also cells in tumor immunity. Int. Immunopharmacol. 34, 244–249, http://dx.doi.
depend on the organ where tumors arise, further complicating the org/10.1016/j.intimp.2016.03.009.
Chiaravalli, A.M., Feltri, M., Bertolini, V., Bagnoli, E., Furlan, D., Cerutti, R., et al.,
complexity of the scenario. Different roles can be played by the
2005. Intratumour T. cells, their activation status and survival in gastric
same population of immune cells in different stages of the dis- carcinomas characterised for microsatellite instability and Epstein-Barr virus
ease (NK cells, i.e.). The role of the immune system in driving an infection. Virchows Arch. 448, 344–353, http://dx.doi.org/10.1007/s00428-
anti- or pro-tumor response is also dependent on the cytokines and 005-0066-4.
Choi, H.S., Ha, S.Y., Kim, H.-M., Ahn, S.M., Kang, M.-S., Kim, K.-M., et al., 2016. The
chemokines present (Fridman et al., 2012). In addition some treat- prognostic effects of tumor infiltrating regulatory T cells and myeloid derived
ments can also exert immune effects (Zitvogel et al., 2008). These suppressor cells assessed by multicolor flow cytometry in gastric cancer
complex interactions could be further complicated by the network patients. Oncotarget 7, 7940–7951.
Churlaud, G., Pitoiset, F., Jebbawi, F., Lorenzon, R., Bellier, B., Rosenzwajg, M., et al.,
of blood and lymphatic vessels, favoring not only metastatic spread 2015. Human and mouse CD8 + CD25 + FOXP3+ regulatory T cells at steady
but also increasing immune infiltration. Considering the promising state and during interleukin-2 therapy. T Cell Biol 6, 171, http://dx.doi.org/10.
results obtained with immunotherapy, given the potential toxici- 3389/fimmu.2015.00171.
Cutsem, E.V., Cervantes, A., Nordlinger, B., Arnold, D., 2014. Metastatic colorectal
ties and the costs related to the use of these new agents, emerging cancer: ESMO clinical practice guidelines for diagnosis, treatment and
is the need for the identification of predictive biomarkers. The role follow-up. Ann. Oncol. 25, iii1–iii9, http://dx.doi.org/10.1093/annonc/mdu260.
of PD-L1 is controversial, since in some PD-L1 negative tumors Dunn, G.P., Bruce, A.T., Ikeda, H., Old, L.J., Schreiber, R.D., 2002. Cancer
immunoediting: from immunosurveillance to tumor escape. Nat. Immunol. 3,
we observed responses to anti-PD-(L)1 agents, whilst a higher
991–998, http://dx.doi.org/10.1038/ni1102-991.
ORR has been seen in most of PD-L1 positive tumors. Metastatic Eggermont, A.M.M., Chiarion-Sileni, V., Grob, J.-J., Dummer, R., Wolchok, J.D.,
melanoma patients with PD-L1 negative tumors seem to benefit Schmidt, H., et al., 2016. Prolonged survival in stage III melanoma with
ipilimumab adjuvant therapy. N. Engl. J. Med., http://dx.doi.org/10.1056/
more from the combination of PD-1 and CTLA-4 blockade (Larkin
nejmoa1611299.
et al., 2015). Novel predictors of benefit from these therapies are Farazi, P.A., DePinho, R.A., 2006. Hepatocellular carcinoma pathogenesis: from
genomic parameters and MSI-H metastatic CRC might represent genes to environment. Nat. Rev. Cancer. 6, 674–687, http://dx.doi.org/10.1038/
ideal candidates for immunotherapy with ICB. Considering that nrc1934.
Ferris, R.L., Blumenschein, G., Fayette, J., Guigay, J., Colevas, A.D., Licitra, L., et al.,
the presence of TILs has been associated with a good prognosis in 2016. Nivolumab for recurrent squamous-cell carcinoma of the head and neck.
many tumor types including GI tumors, the biological correlation N. Engl. J. Med., http://dx.doi.org/10.1056/nejmoa1602252.
C. Solinas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 106–116 115

Fridman, W.H., Pagès, F., Sautès-Fridman, C., Galon, J., 2012. The immune Hu, W.-H., Miyai, K., Cajas-Monson, L.C., Luo, L., Liu, L., Ramamoorthy, S.L., 2015.
contexture in human tumours: impact on clinical outcome. Nat. Rev. Cancer Tumor-infiltrating CD8+ T lymphocytes associated with clinical outcome in
12, 298–306, http://dx.doi.org/10.1038/nrc3245. anal squamous cell carcinoma. J. Surg. Oncol. 112, 421–426, http://dx.doi.org/
Fukunaga, A., Miyamoto, M., Cho, Y., Murakami, S., Kawarada, Y., Oshikiri, T., et al., 10.1002/jso.23998.
2004. CD8+ tumor-infiltrating lymphocytes together with CD4+ Huang, Y., Liao, H., Zhang, Y., Yuan, R., Wang, F., Gao, Y., et al., 2014. Prognostic
tumor-infiltrating lymphocytes and dendritic cells improve the prognosis of value of tumor-infiltrating foxP3 + T Cells in gastrointestinal cancers: a meta
patients with pancreatic adenocarcinoma. Pancreas 28, e26–31. analysis. PLoS One 9, e94376, http://dx.doi.org/10.1371/journal.pone.0094376.
Gabitass, R.F., Annels, N.E., Stocken, D.D., Pandha, H.A., Middleton, G.W., 2011. Hwang, W.-T., Adams, S.F., Tahirovic, E., Hagemann, I.S., Coukos, G., 2012.
Elevated myeloid-derived suppressor cells in pancreatic, esophageal and Prognostic significance of tumor-infiltrating T cells in ovarian cancer: a
gastric cancer are an independent prognostic factor and are associated with meta-analysis. Gynecol. Oncol. 124, 192–198, http://dx.doi.org/10.1016/j.
significant elevation of the Th2 cytokine interleukin-13. Cancer. Immunol. ygyno.2011.09.039.
Immunother. 60, 1419–1430, http://dx.doi.org/10.1007/s00262-011-1028-0. Ikeguchi, M., Hirooka, Y., 2005. Interleukin-2 gene expression is a new biological
Galon, J., Costes, A., Sanchez-Cabo, F., Kirilovsky, A., Mlecnik, B., Lagorce-Pagès, C., prognostic marker in hepatocellular carcinomas. Onkologie 28, 255–259,
et al., 2006. Type, density, and location of immune cells within human http://dx.doi.org/10.1159/000084695.
colorectal tumors predict clinical outcome. Science 313, 1960–1964, http://dx. Ishii, Y., 2013. Elevated expression of cyclooxygenase-2 and microsomal
doi.org/10.1126/science.1129139. prostaglandin E synthase-1 in primary sclerosing cholangitis: implications for
Galon, J., Pagès, F., Marincola, F.M., Thurin, M., Trinchieri, G., Fox, B.A., et al., 2012. cholangiocarcinogenesis. Int. J. Oncol., http://dx.doi.org/10.3892/ijo.2013.2038.
The immune score as a new possible approach for the classification of cancer. J. Janjigian, Y.Y., Sanchez-Vega, F., Jonsson, P., Tuvy, Y., Bouvier, N., Riches, J., et al.,
Transl. Med. 10, 1, http://dx.doi.org/10.1186/1479-5876-10-1. 2016. Clinical next generation sequencing (NGS) of esophagogastric (EG)
Gao, Q., Qiu, S.-J., Fan, J., Zhou, J., Wang, X.-Y., Xiao, Y.-S., et al., 2007. Intratumoral adenocarcinomas identifies distinct molecular signatures of response to HER2
balance of regulatory and cytotoxic t cells is associated with prognosis of inhibition, first-line 5FU/platinum and PD1/CTLA4 blockade. Ann. Oncol. 27,
hepatocellular carcinoma after resection. J. Clin. Oncol. 25, 2586–2593, http:// 612O, http://dx.doi.org/10.1093/annonc/mdw371.04.
dx.doi.org/10.1200/JCO.2006.09.4565. Kang, B.W., Seo, A.N., Yoon, S., Bae, H.I., Jeon, S.W., Kwon, O.K., et al., 2016.
Garnelo, M., Tan, A., Her, Z., Yeong, J., Lim, C.J., Chen, J., et al., 2015. Interaction Prognostic value of tumor-infiltrating lymphocytes in Epstein-Barr
between tumour-infiltrating B cells and T cells controls the progression of virus-associated gastric cancer. Ann. Oncol. 27, 494–501, http://dx.doi.org/10.
hepatocellular carcinoma. Gut, http://dx.doi.org/10.1136/gutjnl-2015-310814, 1093/annonc/mdv610.
gutjnl-2015-310814. Kim, J.W., Nam, K.H., Ahn, S.-H., Park, D.J., Kim, H.-H., Kim, S.H., et al., 2014.
Giampieri, R., Maccaroni, E., Mandolesi, A., Prete, M.D., Andrikou, K., Faloppi, L., Prognostic implications of immunosuppressive protein expression in tumors
et al., 2016. Mismatch repair deficiency may affect clinical outcome through as well as immune cell infiltration within the tumor microenvironment in
immune response activation in metastatic gastric cancer patients receiving gastric cancer. Gastric. Cancer. 19, 42–52, http://dx.doi.org/10.1007/s10120-
first-line chemotherapy. Gastric Cancer 2016, 1–8, http://dx.doi.org/10.1007/ 014-0440-5.
s10120-016-0594-4. Kim, S.Y., Park, C., Kim, H.-J., Park, J., Hwang, J., Kim, J.-I., et al., 2015. Deregulation
Giannakis, M., Mu, X.J., Shukla, S.A., Qian, Z.R., Cohen, O., Nishihara, R., et al., 2016. of immune response genes in patients with epstein-barr virus-associated
Genomic correlates of immune-cell infiltrates in colorectal carcinoma. Cell gastric cancer and outcomes. Gastroenterology 148, 137–147, http://dx.doi.
Rep. 15, 857–865, http://dx.doi.org/10.1016/j.celrep.2016.03.075. org/10.1053/j.gastro.2014.09.020, e9.
Gilbert, D.C., Serup-Hansen, E., Linnemann, D., Høgdall, E., Bailey, C., Summers, J., Kim, J., Bae, J.-S., Kim, J., Bae, J.-S., 2016. Tumor-associated macrophages and
et al., 2016. Tumour-infiltrating lymphocyte scores effectively stratify neutrophils in tumor microenvironment. Mediat. Inflamm. Mediat. Inflamm.
outcomes over and above p16 post chemo-radiotherapy in anal cancer. Br. J. 2016, http://dx.doi.org/10.1155/2016/6058147, 2016:e6058147.
Cancer 114, 134–137, http://dx.doi.org/10.1038/bjc.2015.448. Kumar, V., Patel, S., Tcyganov, E., Gabrilovich, D.I., 2016. The nature of
Glaire, M.A., Domingo, E., Vermeulen, L., Wezel van, T., Liefers, G.-J., Lothe, R.A., myeloid-derived suppressor cells in the tumor microenvironment. Trends
et al., 2016. POLE proofreading domain mutation defines a subset of Immunol. 37, 208–220, http://dx.doi.org/10.1016/j.it.2016.01.004.
immunogenic colorectal cancers with excellent prognosis. Ann. Oncol. 27, Ladoire, S., Martin, F., Ghiringhelli, F., 2011. Prognostic role of FOXP3+ regulatory T
http://dx.doi.org/10.1093/annonc/mdw370.09, 460O. cells infiltrating human carcinomas: the paradox of colorectal cancer. Cancer
Goeppert, B., Frauenschuh, L., Zucknick, M., Stenzinger, A., Andrulis, M., Klauschen, Immunol. Immunother. 60, 909–918, http://dx.doi.org/10.1007/s00262-011-
F., et al., 2013. Prognostic impact of tumour-infiltrating immune cells on biliary 1046-y.
tract cancer. Br. J. Cancer 109, 2665–2674, http://dx.doi.org/10.1038/bjc.2013. Lal, N., Beggs, A.D., Willcox, B.E., Middleton, G.W., 2015. An immunogenomic
610. stratification of colorectal cancer: Implications for development of targeted
Gorosito Serrán, M., Fiocca Vernengo, F., Beccaria, C.G., Acosta Rodriguez, E.V., immunotherapy. OncoImmunology 4, e976052, http://dx.doi.org/10.4161/
Montes, C.L., Gruppi, A., 2015. The regulatory role of B cells in autoimmunity, 2162402X.2014.76052.
infections and cancer: perspectives beyond IL10 production. FEBS Lett. 589, Larkin, J., Chiarion-Sileni, V., Gonzalez, R., Grob, J.J., Cowey, C.L., Lao, C.D., et al.,
3362–3369, http://dx.doi.org/10.1016/j.febslet.2015.08.048. 2015. Combined nivolumab and ipilimumab or monotherapy in untreated
Grabenbauer, G.G., Lahmer, G., Distel, L., Niedobitek, G., 2006. Tumor-infiltrating melanoma. N. Engl. J. Med. 373, 23–34, http://dx.doi.org/10.1056/
cytotoxic T. cells but not regulatory cells predict outcome in anal squamous NEJMoa1504030.
cell carcinoma. Clin. Cancer Res. 12, 3355–3360, http://dx.doi.org/10.1158/ Le, D.T., Uram, J.N., Wang, H., Bartlett, B.R., Kemberling, H., Eyring, A.D., et al., 2015.
1078-0432.CCR-05-2434. PD-1 Blockade in tumors with mismatch-repair deficiency. N. Engl. J. Med. 372,
Gregory, A.D., Houghton, A.M., 2011. Tumor-associated neutrophils new targets for 2509–2520, http://dx.doi.org/10.1056/NEJMoa1500596.
cancer therapy. Cancer Res. 71, 2411–2416, http://dx.doi.org/10.1158/0008- Li, K., Zhu, Z., Luo, J., Fang, J., Zhou, H., Hu, M., et al., 2015. Impact of chemokine
5472, CAN-10-2583. receptor CXCR3 on tumor-infiltrating lymphocyte recruitment associated with
Grivennikov, S.I., Greten, F.R., Karin, M., 2010. IMmunity, inflammation, and cancer. favorable prognosis in advanced gastric cancer. Int. J. Clin. Exp. Pathol. 8,
Cell 140, 883–899, http://dx.doi.org/10.1016/j.cell.2010.01.025. 14725–14732.
Guinney, J., Dienstmann, R., Wang, X., de Reyniès, A., Schlicker, A., Soneson, C., Lin, G., Liu, Y., Li, S., Mao, Y., Wang, J., Shuang, Z., et al., 2016. Elevated
et al., 2015. The consensus molecular subtypes of colorectal cancer. Nat. Med. neutrophil-to-lymphocyte ratio is an independent poor prognostic factor in
21, 1350–1356, http://dx.doi.org/10.1038/nm.3967. patients with intrahepatic cholangiocarcinoma. Oncotarget 5.
Haas, M., Dimmler, A., Hohenberger, W., Grabenbauer, G.G., Niedobitek, G., Distel, Liu, K., Yang, K., Wu, B., Chen, H., Chen, X., Chen, X., et al., 2015. Tumor-infiltrating
L.V., 2009. Stromal regulatory T-cells are associated with a favourable immune cells are associated with prognosis of gastric cancer. Medicine
prognosis in gastric cancer of the cardia. BMC Gastroentero.l 9, 65, http://dx. (Baltimore) 94, http://dx.doi.org/10.1097/MD.0000000000001631.
doi.org/10.1186/1471-230X-9-65. Llovet, J.M., Villanueva, A., Lachenmayer, A., Finn, R.S., 2015. Advances in targeted
Hadjipanayis, Angela, 2014. The cancer genome atlas research network therapies for hepatocellular carcinoma in the genomic era. Nat. Rev. Clin.
comprehensive molecular characterization of gastric adenocarcinoma. Nature Oncol. 12, 436, http://dx.doi.org/10.1038/nrclinonc.2015.121.
513, 202–209, http://dx.doi.org/10.1038/nature13480. Lutz, E.R., Wu, A.A., Bigelow, E., Sharma, R., Mo, G., Soares, K., et al., 2014.
Hiraoka, N., Ino, Y., Yamazaki-Itoh, R., Kanai, Y., Kosuge, T., Shimada, K., 2015. Immunotherapy converts nonimmunogenic pancreatic tumors into
Intratumoral tertiary lymphoid organ is a favourable prognosticator in patients immunogenic foci of immune regulation. Cancer Immunol. Res 2, 616–631,
with pancreatic cancer. Br. J. Cancer 112, 1782–1790, http://dx.doi.org/10. http://dx.doi.org/10.1158/2326-6066.CIR-14-0027.
1038/bjc.2015.145. McGranahan, N., Furness, A.J.S., Rosenthal, R., Ramskov, S., Lyngaa, R., Saini, S.K.,
Hoff, D.D.V., Ramanathan, R.K., Borad, M.J., Laheru, D.A., Smith, L.S., Wood, T.E., et al., 2016. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to
et al., 2011. Gemcitabine plus nab-paclitaxel is an active regimen in patients immune checkpoint blockade. Science 351, 1463–1469, http://dx.doi.org/10.
with advanced pancreatic cancer: a phase I/II trial. J Clin Oncol 29, 4548–4554, 1126/science.aaf1490.
http://dx.doi.org/10.1200/JCO.2011.36.5742. Melero, I., Sangro, B., Yau, T., Hsu, C., Kudo, M., Crocenzi, T., et al., 2016. Safety and
Hou, J., Yu, Z., Xiang, R., Li, C., Wang, L., Chen, S., et al., 2014. Correlation between preliminary efficacy of nivolumab (nivo) in patients (pts) with advanced
infiltration of FOXP3+ regulatory T cells and expression of B7-H1 in the tumor hepatocellular carcinoma (aHCC): Interim analysis of the phase 1/2
tissues of gastric cancer. Exp. Mol. Pathol. 96, 284–291, http://dx.doi.org/10. CheckMate-040 study. Ann. Oncol. 27, 615O, http://dx.doi.org/10.1093/
1016/j.yexmp.2014.03.005. annonc/mdw371.07.
Hu, M., Li, K., Maskey, N., Xu, Z., Peng, C., Wang, B., et al., 2014. Decreased Mlecnik, B., Bindea, G., Angell, H.K., Maby, P., Angelova, M., Tougeron, D., et al.,
intratumoral Foxp3 Tregs and increased dendritic cell density by neoadjuvant 2016. Integrative analyses of colorectal cancer show immunoscore is a
chemotherapy associated with favorable prognosis in advanced gastric cancer. stronger predictor of patient survival than microsatellite instability. Immunity
Int. J. Clin. Exp. Pathol. 7, 4685–4694. 44, 698–711, http://dx.doi.org/10.1016/j.immuni.2016.02.025.
116 C. Solinas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 106–116

Morris, M., Platell, C., Iacopetta, B., 2008a. Tumor-infiltrating lymphocytes and chemoradiotherapy for rectal cancer. Ann. Surg. Oncol. 21, 414–421, http://dx.
perforation in colon cancer predict positive response to 5-fluorouracil doi.org/10.1245/s10434-014-3584-y.
chemotherapy. Clin. Cancer Res. 14, 1413–1417, http://dx.doi.org/10.1158/ Shirabe, K., Matsumata, T., Maeda, T., Sadanaga, N., Kuwano, H., Sugimachi, K.,
1078-0432.CCR-07-1994. 1995. A long-term surviving patient with hepatocellular carcinoma including
Morris, M., Platell, C., de Boer, B., McCaul, K., Iacopetta, B., 2008b. Population-based lymphocytes infiltration–a clinicopathological study. Hepatogastroenterology
study of prognostic factors in stage II colonic cancer. Br. J. Surg. 93, 866–871, 42, 996–1001.
http://dx.doi.org/10.1002/bjs.5345. Siliņa, K., Rulle, U., Kalniņa, Z., Linē, A., 2014. Manipulation of tumour-infiltrating B
Motzer, R.J., Escudier, B., McDermott, D.F., George, S., Hammers, H.J., Srinivas, S., cells and tertiary lymphoid structures: a novel anti-cancer treatment avenue?
et al., 2015. Nivolumab versus everolimus in advanced renal-cell carcinoma. N. Cancer Immunol. Immunother. 63, 643–662, http://dx.doi.org/10.1007/
Engl. J. Med. 373, 1803–1813, http://dx.doi.org/10.1056/NEJMoa1510665. s00262-014-1544-9.
NCCN, 2017. Clinical Practice Guidelines in Oncology n.d (accessed May 21, 2016) Snyder, A., Makarov, V., Merghoub, T., Yuan, J., Zaretsky, J.M., Desrichard, A., et al.,
https://www.nccn.org/professionals/physician gls/f guidelines nojava.asp. 2014. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N.
Naito, Y., Saito, K., Shiiba, K., Ohuchi, A., Saigenji, K., Nagura, H., et al., 1998. CD8+ T Engl. J. Med. 371, 2189–2199, http://dx.doi.org/10.1056/NEJMoa1406498.
cells infiltrated within cancer cell nests as a prognostic factor in human Thomas, N.E., Busam, K.J., From, L., Kricker, A., Armstrong, B.K., Anton-Culver, H.,
colorectal cancer. Cancer Res. 58, 3491–3494. et al., 2013. Tumor-infiltrating lymphocyte grade in primary melanomas is
Ng, S.S.W., Tsao, M.-S., Nicklee, T., Hedley, D.W., 2002. Effects of the epidermal independently associated with melanoma-specific survival in the
growth factor receptor inhibitor OSI-774, tarceva, on downstream signaling population-based genes, environment and melanoma Study. J. Clin. Oncol. 31,
pathways and apoptosis in human pancreatic adenocarcinoma. Mol. Cancer 4252–4259, http://dx.doi.org/10.1200/JCO. 2013.51.3002.
Ther. 1, 777–778, 1 Supported by the National Cancer Institute of Canada and Tumeh, P.C., Harview, C.L., Yearley, J.H., Shintaku, I.P., Taylor, E.J.M., Robert, L.,
the Pat Myhal Fund for Pancreatic Cancer Research 1. et al., 2014. PD-1 blockade induces responses by inhibiting adaptive immune
Niccolai, E., Taddei, A., Ricci, F., Rolla, S., D’Elios, M.M., Benagiano, M., et al., 2016. resistance. Nature 515, 568–571, http://dx.doi.org/10.1038/nature13954.
Intra-tumoral IFN-(-producing Th22 cells correlate with TNM staging and the Unitt, E., Rushbrook, S.M., Marshall, A., Davies, S., Gibbs, P., Morris, L.S., et al., 2005.
worst outcomes in pancreatic cancer. Clin. Sci. 130, 247–258, http://dx.doi.org/ Compromised lymphocytes infiltrate hepatocellular carcinoma: the role of
10.1042/CS20150437. T-regulatory cells. Hepatology 41, 722–730, http://dx.doi.org/10.1002/hep.
Oshikiri, T., Miyamoto, M., Shichinohe, T., Suzuoki, M., Hiraoka, K., Nakakubo, Y., 20644.
et al., 2003. Prognostic value of intratumoral CD8+ T lymphocyte in Vivier, E., Ugolini, S., Blaise, D., Chabannon, C., Brossay, L., 2012. Targeting natural
extrahepatic bile duct carcinoma as essential immune response. J. Surg. Oncol. killer cells and natural killer T cells in cancer. Nat. Rev. Immunol. 12, 239–252,
84, 224–228, http://dx.doi.org/10.1002/jso.10321. http://dx.doi.org/10.1038/nri3174.
Overman, M.J., Kopetz, S., McDermott, R.S., Leach, J., Lonardi, S., Lenz, H.-J., et al., Volovitz, I., Melzer, S., Amar, S., Bocsi, J., Bloch, M., Efroni, S., et al., 2016. Dendritic
2016. Nivolumab ± ipilimumab in treatment (tx) of patients (pts) with cells in the context of human tumors: biology and experimental tools. Int. Rev.
metastatic colorectal cancer (mCRC) with and without high microsatellite Immunol. 35, 116–135, http://dx.doi.org/10.3109/08830185.2015.1096935.
instability (MSI-H): CheckMate -142 interim results. J. Clin. Oncol. 34. WHO, 2016. Cancer. WHO, n.d. http://www.who.int/mediacentre/factsheets/fs297/
Pagès, F., Kirilovsky, A., Mlecnik, B., Asslaber, M., Tosolini, M., Bindea, G., et al., en/(accessed 2016).
2009. In situ cytotoxic and memory T cells predict outcome in patients with Wada, Y., Nakashima, O., Kutami, R., Yamamoto, O., Kojiro, M., 1998.
early-stage colorectal cancer. J. Clin. Oncol. 27, 5944–5951, http://dx.doi.org/ Clinicopathological study on hepatocellular carcinoma with lymphocytic
10.1200/JCO. 2008.19.6147. infiltration. Hepatology 27, 407–414, http://dx.doi.org/10.1002/hep.
Prall, F., Dührkop, T., Weirich, V., Ostwald, C., Lenz, P., Nizze, H., et al., 2004. 510270214.
Prognostic role of CD8+ tumor-infiltrating lymphocytes in stage III colorectal Wang, D., Liu, Y., Gu, Y., Qin, Y., Ji, H., Wu, L., et al., 2015a. Increased number of
cancer with and without microsatellite instability. Hum. Pathol. 35, 808–816, forkhead box P3+ tumor-infiltrating lymphocytes correlates with high
http://dx.doi.org/10.1016/j.humpath.2004.01.022. preoperative albumin level and better survival in patients with stage II or III
Reck, M., Rodríguez-Abreu, D., Robinson, A.G., Hui, R., Cs"oszi, T., Fülöp, A., et al., colorectal cancer. Tumor. Biol. 36, 5407–5414, http://dx.doi.org/10.1007/
2016. Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell s13277-015-3206-8.
lung cancer. N. Engl. J. Med., http://dx.doi.org/10.1056/NEJMoa1606774. Wang, L., Zhai, Z.-W., Ji, D.-B., Li, Z.-W., Gu, J., 2015b. Prognostic value of CD45RO(+)
Reissfelder, C., Stamova, S., Gossmann, C., Braun, M., Bonertz, A., Walliczek, U., tumor-infiltrating lymphocytes for locally advanced rectal cancer following
et al., 2015. Tumor-specific cytotoxic T lymphocyte activity determines 30 Gy/10f neoadjuvant radiotherapy. Int. J. Colorectal Dis. 30, 753–760, http://
colorectal cancer patient prognosis. J. Clin. Invest. 125, 739–751, http://dx.doi. dx.doi.org/10.1007/s00384-015-2226-6.
org/10.1172/JCI74894. Wang, Y., Liu, T., Tang, W., Deng, B., Chen, Y., Zhu, J., Hepatocellular Carcinoma Cells
Rizvi, N.A., Hellmann, M.D., Snyder, A., Kvistborg, P., Makarov, V., Havel, J.J., et al., Induce Regulatory, T., et al., 2016. Cells and lead to poor prognosis via
2015. Mutational landscape determines sensitivity to PD-1 blockade in production of transforming growth factor-?1. Cell Physiol. Biochem. 38,
non-small cell lung cancer. Science 348, 124–128, http://dx.doi.org/10.1126/ 306–318, http://dx.doi.org/10.1159/000438631.
science.aaa1348. Yamashita, Y.-I., Taketomi, A., Morita, K., Fukuhara, T., Ueda, S., Sanefuji, K., et al.,
Rosenberg, J.E., Hoffman-Censits, J., Powles, T., van der Heijden, M.S., Balar, A.V., 2008. The impact of surgical treatment and poor prognostic factors for patients
Necchi, A., et al., 2016. Atezolizumab in patients with locally advanced and with intrahepatic cholangiocarcinoma: retrospective analysis of 60 patients.
metastatic urothelial carcinoma who have progressed following treatment Anticancer Res. 28, 2353–2359.
with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial. Ye, Y., Zhou, L., Xie, X., Jiang, G., Xie, H., Zheng, S., 2009. Interaction of B7-H1 on
Lancet 387, 1909–1920, http://dx.doi.org/10.1016/S0140-6736(16)00561-4. intrahepatic cholangiocarcinoma cells with PD-1 on tumor-infiltrating T cells
Salgado, R., Denkert, C., Demaria, S., Sirtaine, N., Klauschen, F., Pruneri, G., et al., as a mechanism of immune evasion. J. Surg. Oncol. 100, 500–504, http://dx.doi.
2015. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: org/10.1002/jso.21376.
recommendations by an International TILs Working Group 2014. Ann. Oncol. Zeng, D.-Q., Yu, Y.-F., Ou, Q.-Y., Li, X.-Y., Zhong, R.-Z., Xie, C.-M., et al., 2016.
26, 259–271, http://dx.doi.org/10.1093/annonc/mdu450. Prognostic and predictive value of tumor-infiltrating lymphocytes for clinical
Sangro, B., Gomez-Martin, C., de la Mata, M., Iñarrairaegui, M., Garralda, E., Barrera, therapeutic research in patients with non-small cell lung cancer. Oncotarget,
P., et al., 2013. A clinical trial of CTLA-4 blockade with tremelimumab in http://dx.doi.org/10.18632/oncotarget.7282.
patients with hepatocellular carcinoma and chronic hepatitis C. J. Hepatol. 59, Zhang, L., Qiu, M., Jin, Y., Ji, J., Li, B., Wang, X., et al., 2015. Programmed cell death
81–88, http://dx.doi.org/10.1016/j.jhep.2013.02.022. ligand 1 (PD-L1) expression on gastric cancer and its relationship with
Shao, Y., Lo, C.M., Ling, C.C., Liu, X.B., Ng, K.T.-P., Chu, A.C.Y., Regulatory, B., et al., clinicopathologic factors. Int. J. Clin. Exp. Pathol. 8, 11084–11091.
2014. cells accelerate hepatocellular carcinoma progression via CD40/CD154 Zhou, S., Xu, S., Tao, H., Zhen, Z., Chen, G., Zhang, Z., et al., 2013. CCR7 Expression
signaling pathway. Cancer Lett. 355, 264–272, http://dx.doi.org/10.1016/j. and Intratumoral FOXP3 + Regulatory T Cells are Correlated with Overall
canlet.2014.09.026. Survival and Lymph Node Metastasis in Gastric Cancer. PLoS One 8, e74430,
Shen, Z., Zhou, S., Wang, Y., Li, R., Zhong, C., Liang, C., et al., 2010. Higher http://dx.doi.org/10.1371/journal.pone.0074430.
intratumoral infiltrated Foxp3+ Treg numbers and Foxp3+/CD8+ ratio are Zitvogel, L., Tesniere, A., Apetoh, L., Ghiringhelli, F., Kroemer, G., 2008.
associated with adverse prognosis in resectable gastric cancer. J. Cancer Res. Immunological aspects of anticancer chemotherapy. Bull. Académie Natl. Méd.
Clin. Oncol. 136, 1585–1595, http://dx.doi.org/10.1007/s00432-010-0816-9. 192, 1469-1487-1489.
Shinto, E., Hase, K., Hashiguchi, Y., Sekizawa, A., Ueno, H., Shikina, A., et al., 2014.
CD8+ and FOXP3+ Tumor-infiltrating T. cells before and after