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
1. Introduction
The interaction between neoplastic cells and the immune sys-
tem starts during the early phases of tumor development, when
strongly immunogenic cells are recognized and eliminated by
the immune response. In the equilibrium and escape phases, the
most aggressive and less immunogenic cells are able to survive
and finally evade immunity, tumor becoming clinically manifested
(Dunn et al., 2002). The presence of an extensive immune infil-
trate has been associated with improved patients outcomes (better
survival and responses to treatments) in a variety of solid tumors
[ovarian, colorectal (CRC), non-small cell lung cancer (NSCLC),
melanoma, HER2-positive and triple-negative (TN) breast cancer
(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-
tion of new immunotherapeutic agents, the immune checkpoint
inhibitors, showed promising results in the treatment of several
advanced neoplasms, like melanoma, kidney, NSCLC, bladder can-
cer and head and neck squamous cell carcinomas, giving rise to
durable responses also in heavily pre-treated patients (Reck et al.,
2016; Ferris et al., 2016; Larkin et al., 2015; Motzer et al., 2015;
Borghaei et al., 2015; Rosenberg et al., 2016). In the adjuvant set-
ting, higher rates of recurrence-free survival (RFS), overall survival
(OS) and distant metastasis-free survival were observed in stage
III melanoma patients treated with the anti-CTLA-4 ipilimumab
(Eggermont et al., 2016). Nevertheless the activity of these drugs
is limited to a proportion of subjects and is associated with non-
negligible side effects, leading to a strong need for the identification
of the best responders to these treatments.
1.1. The elements of the tumor immune environment: from
innate to adaptive immunity
The tumor immune environment can be oriented towards
an anti-tumor or alternatively to an inflammatory pro-tumor
response, depending on the (dynamic) balance of the different
subpopulations of its tumor infiltrating leukocytes and their func-
tional status. For example, tumor associated neutrophils (TAN)
and macrophages (TAM) can be polarized in different popula-
tions playing opposite roles, anti- (N1 and M1) and pro-tumor (N2
and M2) respectively (Gregory and Houghton, 2011; Kim et al.,
2016). Myeloid derived suppressor cells (MDSC) are immature
myeloid cells that dampen T cell activation in a not antigen (Ag)
specific way, through different mechanisms (release of inhibitory
molecules and deprivation of amino acids involved in the prolif-
eration of lymphocytes) (Kumar et al., 2016). Dendritic cells (DC)
are professional Ag presenting cells (APC) that migrate in and
out of tissues and lymph-nodes; some DC subpopulations can be
immune-suppressive, others are not (Volovitz et al., 2016). Natu-
ral killer (NK) cells target those cells that have lost the expression
of MHC proteins (the so called missing-self) and can be also acti-
vated by stressed-induced ligands that are upregulated by tumor
cells (namely the stress-induced self). Additionally, NK cells can
shape the tumor microenvironment by producing cytokines that
are involved in the differentiation, activation and recruitment of
other immune cells (Vivier et al., 2012). In the context of adap-
tive immunity, T and B lymphocytes drive an Ag specific response,
through T cell (TCR) and B cell receptors (BCR) respectively. T lym-
phocytes are differentiated in various subpopulations with distinct
profiles: some subsets of CD4+ helper T cells can have anti- (Th1,
Tfh, . . .), pro-tumor (i.e.: Th2) or regulatory functions (Treg); cyto-
toxic CD8+ T lymphocytes (CTL) mostly kill tumor cells and release
cytokines (Chen et al., 2016). B lymphocytes can present Ags, pro-
duce antibodies and cytokines and also behave as regulatory cells
(Breg), inhibiting the immune response (Siliņa et al., 2014; Gorosito
Serrán et al., 2015) (Fig. 1).
107
Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells exert
a cytotoxic activity. Tumor associated macrophages (TAM) and
tumor associated neutrophils (TAN) can play both anti-tumor or
pro-tumor roles depending on their polarization (M1 and N1 or
M2 and N2 respectively). Dendritic cells (DC) and B lymphocytes
are antigen presenting cells (APC). B cells can also produce anti-
bodies (Ab), secrete cytokines and behave as regulatory cells. T
regulatory lymphocytes (Treg) secrete soluble factors (IL-10 and
TGF-␤, i.e.) and express high levels of the inhibitory immune check-
point molecule CTLA-4 playing a suppressive role and regulating
the activity of some other immune cells at the tumor site.
1.2. The assessment of the immune infiltrate in tumors
In order to investigate the clinical significance of the immune
infiltrate in tumors, TILs evaluation has been done with various
methods: gene signatures (analyzing the mRNA expression of spe-
cific cytokines and genes involved in the immune response, with
activity and functional information) and pathological assessment
on hematoxylin-eosin (H&E), immunohistochemistry (IHC) and
immunofluorescence (IF) stained slides. H&E colored tumor sec-
tions are routinely used, and allow the evaluation of the extent of
mononuclear inflammatory cells infiltration, which has a prognos-
tic and predictive significance in TN and HER2-positive BC, i.e. The
use of specific antibodies at IHC and IF (i.e.: anti-CD3, −CD20, −CD4,
−CD8, markers for T, B, CD4+ and CD8+ T lymphocytes respectively)
accurately identifies the various subpopulations of TILs (Salgado
et al., 2015). Microscopic analysis gives information on TILs spatial
distribution (at the invasive margin, in the stroma or within cancer
cell nests) and density that have been shown to be prognostic in
CRC (Galon et al., 2006).
1.3. TILs and benefit from immunotherapy
TILs have also been studied as predictive markers of response to
the new immune checkpoint blockade (ICB) therapies. Metastatic
melanoma patients who benefit more from the anti-PD-1 pem-
brolizumab had high extent of proliferating CD8+ TILs at the tumor
invasive margin, PD-L1+, PD-1+ immune cells and a more clonal
TILs TCR repertoire (Tumeh et al., 2014). The link between tumor
genomics and better responses to immunotherapy has also been
investigated in metastatic melanoma patients treated with the
CTLA-4 blockade, where a high mutational load and the pres-
ence of specific neo-Ags predicted major clinical benefit from
the treatment (Snyder et al., 2014). In NSCLC objective responses
(OR), durable clinical benefit, and progression-free survival (PFS)
improved during treatment with pembrolizumab. These responses
were seen in the presence of an inflamed microenvironment
together with activated effector T cells, in the presence of clonal
neo-Ag and a high nonsynonymous mutation burden (McGranahan
et al., 2016; Rizvi et al., 2015). Pembrolizumab administered in
first line in advanced NSCLC patients with tumors expressing PD-
L1 on at least 50% of neoplastic cells gave rise to longer PFS
and overall survival (OS) with respect to standard platinum-based
chemotherapy (Reck et al., 2016). In metastatic CRC patients the
mismatch repair (MMR) deficiency status predicted benefit from
the anti-PD-1 treatment. This condition, caused by the inactiva-
tion of MMR-associated genes following either epigenetic silencing
or germline mutations (with an additional somatic inactivation of
the second allele) generates a large number of somatic mutations
(thousands versus hundreds that are found in mismatch-proficient
tumors). These mutations accumulate in regions of repetitive DNA,
leading to microsatellite instability (MSI). In patients treated with
pembrolizumab, IHC expression of CD8 and PD-L1 by TILs was not
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
clinical activity of pembrolizumab has been investigated in a phase
II trial evaluating the treatment of metastatic MSI-H and MSS
CRC patients. Their results showed that MMR–deficient (MSI-H)
are more responsive to PD-1 blockade than MMR–proficient
(MSS) tumors. This can be explained by the increased number
of mutation-associated neo-Ags resulting from MMR deficiency
driving an higher immune-infiltration (Le et al., 2015). In this group
of metastatic MSI-H patients the combination of the anti-PD-1
nivolumab with the anti-CTLA-4 ipilimumab has been tested in
the CheckMate-142 trial. Partial responses (PR) were observed in
25.5% and 33.3% of the patients from the anti-PD-1 alone and the
combination arms respectively (Overman et al., 2016). Neverthe-
less even within MSS tumors, an increased overall lymphocytic
score has been linked with higher neo-Ag load and a better CRC-
specific survival (at both univariate and multivariate analyses).
Among MSS, higher neo-Ag load and a higher degree of TILs were
observed in polymerase epsilon (POLE)-mutated cases, suggesting
that this genomic alteration, generating “ultra-mutated” tumors
could be determinant for immune infiltration (Giannakis et al.,
2016). Pathogenic POLE-proofreading domain mutations occur
in 1-10% of CRC and are more frequent in younger with a 7.6%
prevalence in patients aged <40 years old. These POLE-mutated
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
outcome observed particularly in stage II disease (Glaire et al.,
2016). ICB therapeutic approach is underway in this group of
patients.
While MSI-H and POLE mutations in CRC have been associated
with high mutational burden and high TILs, RAS mutant tumors
usually have a poor immune infiltration and a lower expression of
immune checkpoint molecules, indicating that these mutations are
probably less immunogenic (Lal et al., 2015).
Conversion to an inflamed phenotype in MSS colon cancers can
be achieved through the inhibition of mitogen-activated protein
kinase (MAPK) pathway together with a PD-L1 blockade, as shown
in a phase Ib study testing the combination of cobimetinib and
atezolizumab. Higher CD8+ and MHC-I expression by tumor cells
resulted from a serial biopsy cohort in patients receiving these two
drugs (Bendell et al., 2016).
TILs have also been shown to have a predictive value in rec-
tal cancers receiving a neo-adjuvant treatment. High CD8+/Foxp3+
intraepithelial ratio before chemo-radiotherapy (CRT) predicted
tumor regression. High CD8+ stromal density after the treat-
ment was associated with a favorable clinical 5-year recurrence
free survival (RFS). After CRT, stromal CD8+ TILs density doubled,
whereas stromal Foxp3+ counts remained stable, demonstrating
that treatments may influence the composition of TILs subpopula-
tions (Shinto et al., 2014) (Table 1).
Further predictor of tumor down-staging, of a better response
after pre-operative RT and of a longer DFS (at the univariate and
multivariate analyses) was a high density of CD45RO+ TILs, with
several studies confirming these findings (Morris et al., 2008a;
Morris et al., 2008b; Prall et al., 2004; Wang et al., 2015b) (Table 1).
2.2. Anal cancer
Anal tumors, including squamous cell carcinomas (SCC, the most
common), adenocarcinomas, basal cells carcinomas, melanomas
and GIST account for around 2–3% of GI cancers. Around 90%
of anal cancers are attributable to human papillomavirus (HPV)
infection and further risk factors for the development of this dis-
ease are linked to immune suppression and autoimmune disorders
(NCCN, 2017). A study evaluating a variety of TILs subpopulations
(CD3+, CD4+, CD8+ and CD68 + ) in biopsies from 38 patients with
anal carcinoma showed that CD3+ and CD4+ TILs and the expres-
sion of GZMB at the tumor site were negative prognostic factors
(Grabenbauer et al., 2006). Lately, high levels of peri- and intra-
tumoral CD8+ TILs in 40 patients diagnosed with SCC showed a
favorable effect on OS (Hu et al., 2015) (Table 1). Foxp3+ TILs did
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+/−
chemotherapy (CT) for non-metastatic SCC of the anus and the anal
canal, stratified for HPV status (Gilbert et al., 2016) (Table 1).
3. TILs in gastric cancer
GC is the fifth most common cancer in incidence and is the third
most common type for mortality worldwide (Cancer Res, 2015).
Its etiology is strongly related to chronic infections of Helicobacter
pylori (HP) and Epstein-Barr virus (EBV) and immune regulation
signaling plays a crucial role in its pathogenesis (Prall et al., 2004).
The new molecular classification proposed by The Cancer Genome
Atlas (TCGA) (Hadjipanayis, 2014), identified four GC subtypes:
the EBV infected tumors (EBViGC) showing amplification of CD274
(PD-L1) and of PDCD1LG2 (PD-L2) genes, microsatellite unstable
tumors (MSIGC) with elevated mutation rates, genomically stable
tumors (GSGC) and tumors with chromosomal instability (CINGC).
GC with EBV infection or MSI-H have been shown to have high
TILs infiltration and typically have a better prognosis, demonstrat-
ing the fundamental role played by the host response in this disease
(Chiaravalli et al., 2005; Kim et al., 2015; Kang et al., 2016; Giampieri
et al., 2016) (Table 2). Low density of CD4+ T helper and of CD8+ TILs
in radically resected GC have been correlated with a positive lymph
node status and a higher stage at diagnosis. Improved survival has
been observed in the presence of high density of CD4+, CD8+ and
NK TILs. Independent predictors of a poor OS were high densities of
TAN and TAM (Liu et al., 2015; Shen et al., 2010) (Table 2). Foxp3+
lymphocytes infiltration correlated with PD-L1 expression and the
combined presence of these two markers was associated with a
worse prognosis (Hou et al., 2014) (Table 2). Although the gen-
eral trend of considering CD8+ TILs as favorable and Foxp3+ TILs
as an unfavorable marker in radically resected GC, many studies
showed contrasting conclusions (Choi et al., 2016; Haas et al., 2009)
(Table 2). The role of the chemokine receptors that are involved in
TILs recruitment has also been investigated: chemokine receptor 3
(CXCR3) expression has been associated with increased CD4+/CD8+
infiltration and improved OS (Li et al., 2015), high levels of the
chemokine receptor 7 (CCR7) correlated with a positive nodal sta-
tus and high Foxp3+ infiltration and it was associated with poor
OS (Zhou et al., 2013) (Table 2). Inconclusive results came also
from studies evaluating the prognostic significance of the immune
checkpoint molecule PD-L1 expression in the post-operative set-
ting (Kim et al., 2014; Zhang et al., 2015). CTLA-4 was associated
with better outcomes (Kim et al., 2014). In the neo-adjuvant set-
ting, patients receiving a pre-operative treatment with FOLFOX6
had less intra-tumoral Foxp3+ lymphocytes and more DC infil-
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-
ease positively correlated with the depth of tumor invasion and
low Foxp3+ together with high DC emerged as independent prog-
nostic factors for better OS (Hu et al., 2014) (Table 2). High CD3+
TILs assessed in the primary tumor were positively correlated with
MMR deficiency and represented an independent prognostic factor
for better PFS and OS in a cohort of metastatic patients treated with
a first-line platinum-based chemotherapy. Given their high neo-
antigenic potential, as well as their high TILs, this subset of patients
might represent the best candidates to receive immunotherapy
with the immune checkpoint blockade (Le et al., 2015; Giampieri
et al., 2016). Immunotherapy with anti-PD-1 alone or in combina-
tion with the anti-CTLA-4 showed activity and lasting responses in
metastatic GC patients with MSI-H tumors. A complete response
(CR) was also achieved in one patient with a MSS/EBV+ tumor
receiving the dual blockade (Janjigian et al., 2016).
4. TILs in liver tumors
4.1. Hepatocellular carcinoma
Hepatocellular carcinoma (HCC) accounts for up to 90% of all pri-
mary liver tumors, representing the sixth most common cancer(s)
and the second most common cause of cancer death worldwide,
with its incidence increasing (Cancer Res, 2015). Chronic inflamma-
tion induced by multiple etiological factors, mostly viral infections
(HCV and HBV), alcohol consumption and metabolic syndrome,
plays an important role in the malignant transformation of the hep-
atocytes during the development of HCC and a variety of immune
cells (NK, APC, T and B lymphocytes) and cytokines (TGF-␤, IL-2)
participate in this process (Farazi and DePinho, 2006).
At time of diagnosis, only one third of patients are eligible
for potentially curative therapies including resection, transplan-
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
represent the only available options with mOS of 26 and 11 months
respectively (Llovet et al., 2015).
The presence of intra-tumoral TILs is associated with a better
RFS. TILs were located within the trabeculae of neoplastic cells and
were mostly composed of T lymphocytes (Shirabe et al., 1995). In a
retrospective study of 163 consecutive surgical cases of HCC, only
7% of tumors had high TILs infiltration with a predominant pres-
ence of T lymphocytes (mostly CD8+, followed by CD4+). Patients
whose tumors showed this marked infiltration had significantly
higher OS and lower 5 year recurrence rates with respect to the
TILs negative/low group (Wada et al., 1998) (Table 3).
Similar findings were confirmed for the presence of high intra-
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-
tive study of 112 HCC patients. In this study, TILs subpopulations
evaluation was done by flow-cytometry (on fresh tissues), IHC
and IF (on FFPE tissue blocks) and quantitative polymerase chain
reaction (qPCR) was used for the evaluation of mRNA expression
of CD19, CD8A and IFNG (markers for B, CD8+ lymphocytes and
interferon (IFN)-␥, respectively). B lymphocytes were topographi-
cally close to T cells, and their presence was positively associated
with an increased expression of GZMB and IFN-␥. Furthermore, the
expression of co-stimulatory molecules (CD27, CD40), regulating
functional interactions between B and T cells, were linked to better
OS (Garnelo et al., 2015) (Table 3).
High CD8+ TILs density evaluated by IHC has been positively
correlated with IL-2 expression within a cohort of 59 surgically
resected HCC patients. High mRNA expression of IL-2 was asso-
ciated with high CD8+ T cells infiltration and patients with IL-2
positive tumors (56%) had a significant longer 5-year OS rate. The
expression of IL-2 gene emerged as a prognostic biomarker at both
univariate and multivariate analyses and was independent from
tumor stage (Ikeguchi and Hirooka, 2005) (Table 3).
Treg, identified by the expression of CD4, CD25 (the receptor for
IL-2), CD45RO, and of high levels of intra-cellular CTLA-4 by flow
cytometry represented around 9% of isolated TILs from HCC fresh
tissue samples. Co-culture of Treg isolated from TILs with autol-
ogous circulating lymphocytes showed Treg ability in inhibiting
activity, proliferation, perforin expression and IFN-␥ secretion by
CD8+ T cells (Unitt et al., 2005). Low intra-tumoral Foxp3+ with a
concomitant high expression of GZMB+ by TILs represent indepen-
dent prognostic factors for an improved DFS and OS, confirming
that a balance towards activated CD8+ lymphocytes reflects an anti-
tumor immune response (Gao et al., 2007) (Table 3). Other studies
suggest that a high extent of Treg in association with high levels
of transforming growth factor beta (TGF-␤) correlated with a poor
prognosis (Wang et al., 2016) (Table 3). A study of 75 HCC patients
demonstrated that increased levels of CD19 + CD24 + CD39+ circu-
lating Breg in HCC patients were associated with later UICC stages
and venous infiltration at diagnosis. The presence of B TILs, identi-
fied by IHC on FFPE blocks tissue samples showed that they were
mostly localized at the tumor margin rather than in the center of
the tumor area. B cells infiltration was positively associated with
hepatitis B surface Ag positive status, higher tumor size, venous
infiltration and later UICC stages (Shao et al., 2014) (Table 3).
A variety of immunotherapeutic agents (including DC-based
therapies and ICB) has been tested in advanced HCC. Disease control
with a time to progression of 6.4 months was obtained in patients
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
was administered in patients not amenable to curable resection,
that were intolerant or not previously treated with sorafenib (for
refusal). In the HBV infected cohort an effective anti-HBV therapy
was given. OR were durable and observed at all dose levels and in
all etiologic subtypes (uninfected: sorafenib naïve/intolerant; unin-
fected: sorafenib progressor; HCV and HBV). In the dose expansion
cohort OS rate was of 71% at 9 months and safety profile was similar
to what observed in other tumor types. Of note, PD-L1 expression
did not impact on response to PD-1 blockade (Melero et al., 2016).
4.2. Biliary tract cancers
Biliary tract cancers (BTC), are the second most common type of
primary hepatic neoplasms after HCC. They represent a heteroge-
neous group of tumors, distinguished into: intrahepatic (ICC) and
extrahepatic cholangiocarcinoma (ECC) and gallbladder adenocar-
cinoma (GBAC). The prognosis of these diseases is unfavorable and
diagnosis is often delayed. The majority of patients that undergo
surgery develop recurrence after resection; CT and RT offer limited
benefits and there is a strong need for new alternative treatments
(Yamashita et al., 2008; Cabrera, 2013). One of the most com-
mon risk factors associated with BTC development is the presence
of a pre-existing primary sclerosing cholangitis, which leads to a
chronic inflammation state, with an upregulation of several medi-
ators which are believed to be involved in carcinogenesis and tumor
proliferation (Ishii, 2013). However, as stated before, the immune
environment can also inhibit tumor growth and/or invasion, in the
presence of a particular milieu of cytokines (IFN-␥ i.e.) and in the
presence of some subpopulations of TILs that can exert a more
prominent anti-tumor activity. The presence of CD8+ T lympho-
cytes is linked to an anti-tumor immune response (Oshikiri et al.,
2003) (Table 3). Nevertheless their function could be inhibited by
the interaction between PD-1 (expressed on CD8+ T cells follow-
ing activation) and its ligands (PD-L1 and PD-L2) expressed on the
surface of tumor and immune cells within the microenvironment.
In a retrospective study of 435 BTC cases where TILs have been
evaluated by IHC, the most common immune cells detected at the
tumor site were CD8+, followed by CD4+; Foxp3+ TILs were 12%. B
cells and NK infiltrating TILs were rarely found, whilst macrophages
were frequently close to the tumor epithelium. The amount of lym-
phocytes (CD8+, CD4+, B and NK) was inversely proportional to the
stage of disease, with higher extent observed in dysplastic lesions
and a progressive reduction in their amount observed in more
advanced lesions towards metastases. Interestingly the trend was
different for macrophages whose number increased in invasive and
secondary lesions. Higher extent of CD8+, CD4+, CD20+ and Foxp3+
intraepithelial TILs correlated with a longer OS. TILs impact on the
outcome has been demonstrated for GBAC and ECC, not for ICC
patients. These results suggest that BTC might have important dif-
ferences with regard to density and composition of TILs according
to the organ site of origin (Goeppert et al., 2013) (Table 3). A study of
102 consecutive patients diagnosed with ICC evaluated the prog-
nostic role of the neutrophil-to-lymphocyte ratio (NLR) obtained
by dividing the absolute number of neutrophils by the absolute
count of lymphocytes in peripheral blood. Low NLR was associated
with better outcomes (RFS and OS at the univariate and multivari-
ate analyses) and with a higher CD3+ and CD8+ TILs infiltration
at the tumor site. A higher frequency of CD4+ PD-1+ and of CD8+
PD-1+ TILs was seen in patients with high NLR, suggesting that the
evaluation of NLR in the peripheral blood could reflect the activ-
ity (anti- vs pro-) of the immune infiltrate facing with the tumor
(Lin et al., 2016) (Table 3). A higher expression of PD-L1 by tumor
cells analyzed on 31 surgically resected ICC tumors was associated
with more advanced stages of disease and with a poorer tumor dif-
ferentiation. A large number of PD-1+ T lymphocytes (both CD4+
and CD8+) were observed in the stroma or within cancer nests. A
lower rate of CD8+ TILs was associated with more advanced disease
stages (III–IV) and with a high tumor grade. CD8+ TILs presence was
inversely correlated with the overexpression of PD-L1 (Ye et al.,
2009) (Table 3).
5. TILs in pancreatic cancer
Pancreatic cancer (PC) is the seventh leading cause of cancer
mortality worldwide, representing an aggressive disease with a
poor prognosis (Cancer Res, 2015). This disease is often diagnosed
at advanced stages, and surgery represents a suitable solution for a
minority of patients. The chemotherapeutic drugs and the biologic
agents targeting the EGFR pathway (cetuximab and erlotinib) used
in combination with gemcitabine confer a marginal benefit in terms
of OS (Ng et al., 2002; Hoff et al., 2011). New treatment options
are urgently needed and immunotherapy could represent a valid
opportunity in particular subgroups of PC patients. Four molecu-
lar subtypes of PC have been recently identified: squamous, ADEX,
pancreatic progenitor and immunogenic (associated with muci-
nous non-cystic adenocarcinomas and carcinomas arising from
intra-ductal papillary mucinous neoplasia). Immunogenic subtype
showed high expression of immune gene signatures related to
infiltrating B and T cells (both cytotoxic and regulatory), with
upregulation of CTLA-4 and PD-1 pathways. High expression of
macrophage and T cell co-inhibition signatures in a subgroup of PC
patients was associated with a poorer cumulative OS (Bailey et al.,
2016). These observations highlight the significant role played by
the immune system among the different molecular subtypes of PC.
Infiltration by both CD8+ and CD4+ TILs represented a favorable
prognostic factor after surgery in terms of OS rate. This condition
was also associated with less advanced TNM stages and inferior
tumor depth at diagnosis (Fukunaga et al., 2004) (Table 4). Through
IL-10 and TGF-␤ secretion and CTLA-4 expression, Treg regulate a
variety of immunosuppressive mechanisms favoring tumor pro-
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
survival after PC resection. Treg lymphocytes that are specific for
the ␣-enolase Ag (that can also be expressed by PC cells) inhibit
the recruitment and the activation of the Th1 and Th17 effector T
cells (Amedei et al., 2013). High circulating levels of immunosup-
pressive MDSC and CD4+ CD25+ CD127low Foxp3+ Treg were found
to be elevated in PC cancer patients. MDSC positively correlated
with high Treg levels, representing an independent factor for poor
OS (Gabitass et al., 2011). The expression of intra-tumoral IL-22,
whose major source were IFN-␥ producing and perforin-mediated
cytotoxic T cells therefore named Th22 cells was associated with
more advanced stages at diagnosis and worse OS (Niccolai et al.,
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
analyses) (Hiraoka et al., 2015) (Table 4). An induction of similar
aggregates has been observed after the administration of a vaccine
given with or without cyclophosphamide in the neo-adjuvant set-
ting. After 2 weeks of treatment, patients underwent surgery and
analysis of tumor tissue sections. These showed that the majority
of tumors (85%) had tertiary lymphoid organs and that PD-L1 was
expressed by immune cells in aggregates which were surrounded
by PD-1+ cells. Treg infiltration was also induced by the vaccine and
the balance between effector T cells and Treg was increased after
the treatment (Lutz et al., 2014).
6. Conclusions and perspectives
Chronic inflammation following infections (such as EBV, HCV
and HBV), autoimmune diseases (i.e. inflammatory bowel disease)
or prolonged exposure to environmental irritants (like smoke or
obesity) plays a crucial role in the process of GI cancer develop-
ment. Tumor-associated inflammation enhances neo-angiogenesis,
promotes tumor progression and metastatic spread, causes local
immunosuppression and further augments genomic instability.
Despite its pro-tumorigenic effects, inflammation also influences
the host immune response to tumors and can be manipulated
through cancer immunotherapy (Grivennikov et al., 2010). The
presence of CTL, memory T and Th1 cells has been generally asso-
ciated with better outcomes. The role played by other subsets
of T cells is controversial and it can be dependent on the differ-
ent balance of cells from the immune environment. It might also
depend on the organ where tumors arise, further complicating the
complexity of the scenario. Different roles can be played by the
same population of immune cells in different stages of the dis-
ease (NK cells, i.e.). The role of the immune system in driving an
anti- or pro-tumor response is also dependent on the cytokines and
chemokines present (Fridman et al., 2012). In addition some treat-
ments can also exert immune effects (Zitvogel et al., 2008). These
complex interactions could be further complicated by the network
of blood and lymphatic vessels, favoring not only metastatic spread
but also increasing immune infiltration. Considering the promising
results obtained with immunotherapy, given the potential toxici-
ties and the costs related to the use of these new agents, emerging
is the need for the identification of predictive biomarkers. The role
of PD-L1 is controversial, since in some PD-L1 negative tumors
we observed responses to anti-PD-(L)1 agents, whilst a higher
ORR has been seen in most of PD-L1 positive tumors. Metastatic
melanoma patients with PD-L1 negative tumors seem to benefit
more from the combination of PD-1 and CTLA-4 blockade (Larkin
et al., 2015). Novel predictors of benefit from these therapies are
genomic parameters and MSI-H metastatic CRC might represent
ideal candidates for immunotherapy with ICB. Considering that
the presence of TILs has been associated with a good prognosis in
many tumor types including GI tumors, the biological correlation
between TILs and an active immune response also suggests that this
parameter may also have a predictive role during immunotherapy.
This suggestion, mainly deriving from studies in melanoma patients
seems applicable to GI cancer patients as well. However further
investigations are needed to clarify whether the sole assessment
of TILs or together with the immune gene signatures evaluation
could be used as valid biomarker(s) for the selection of the best
candidates for immunotherapy.
Conflict of interest
The authors declare that they have no conflict of interest.
Funding
This review article did not receive any specific grant from fund-
ing agencies in the public, commercial, or not-for-profit sectors.
Acknowledgement
No writing assistance was utilized in the production of the
manuscript.
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