Tumor-infiltrating lymphocyte subsets and tertiary lymphoid structures in pulmonary metastases from colorectal cancer
Clin Exp Metastasis
Tumor-infiltrating lymphocyte subsets and tertiary lymphoid structures in pulmonary metastases from colorectal cancer
Thomas Schweiger 0 1 2 3 4 5
Anna Sophie Berghoff 0 1 2 3 4 5
Christoph Glogner 0 1 2 3 4 5
Olaf Glueck 0 1 2 3 4 5
Orsolya Rajky 0 1 2 3 4 5
Denise Traxler 0 1 2 3 4 5
Peter Birner 0 1 2 3 4 5
Matthias Preusser 0 1 2 3 4 5
Walter Klepetko 0 1 2 3 4 5
Konrad Hoetzenecker 0 1 2 3 4 5
0 Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Medical University of Vienna , Vienna , Austria
1 Division of Thoracic Surgery, Department of Thoracic Surgery, Medical University of Vienna , Waehringer Guertel 18-20, 1090 Vienna , Austria
2 & Konrad Hoetzenecker
3 Department of Pathology, Medical University of Vienna , Vienna , Austria
4 Comprehensive Cancer Center, Medical University of Vienna , Vienna , Austria
5 Department of Medicine I, Medical University of Vienna , Vienna , Austria
The presence of tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLSs) reflects an active inflammatory tumor microenvironment. High density of TILs as well as presence of TLS is associated with improved survival in various solid cancer types. We aimed to describe the density and distribution of TILs and TLS in pulmonary metastases (PMs) from primary colorectal cancer (CRC) and its correlation with clinicopathological variables. Fifty-seven CRC pulmonary metastasectomy specimen (PM) and 31 matched primary CRC specimen were included. Cluster of differentiation (CD)3?, CD8?, CD45RO? and FoxP3? TILs were evaluated by immunohistochemistry and density was scored semiquantitatively. TLS were evaluated based on morphological criteria. Survival time was defined from pulmonary metastasectomy to death or last follow up. A marked infiltration with CD3?, CD8?, CD45RO? and FoxP3? TILs was evident in CRC PM and matched primary CRC. Further assessment of the immune infiltrate in PM showed that a high density of FOXP3? TILs at the invasive margin [HR 2.40 (1.11-6.96); P = 0.031] and low density of CD8? cells in TLS [HR 0.30 (0.14-0.79); P = 0.016] were associated with a worse prognosis in univariate analysis. Moreover, a low CD8/FoxP3-ratio of TILs at the invasive margin (P = 0.042) and in TLS (P = 0.027) conferred an impaired prognosis after pulmonary metastasectomy. Our findings suggest that CRC PM harbor an immune active microenvironment. The balance of CD8? and FoxP3? T-cells at the tumor border and in TLS provides prognostic information in patients with CRC PM.
Pulmonary metastasis; Colorectal cancer; Tumor-infiltrating lymphocytes; TILs; Tertiary lymphoid structures
Background
Despite advances in the early detection and treatment of
colorectal cancer (CRC), the prognosis of patients is
impaired as soon as distant metastases occur. Synchronous
pulmonary spreading is evident in about one out of ten
patients with newly diagnosed CRC. Subsequently, an
average 5-year cumulative risk of 5.8 % for the development
of metachronous pulmonary metastases (PMs) is
additionally contributing to the disease burden of patients with CRC
[
1
]. However, within the group of patients with CRC lung
metastases long-term survival can be achieved by (repeated)
pulmonary metastasectomy complemented by
chemotherapeutic regimens. In contrast, some patients will present with
diffuse recurrence of disease within months after pulmonary
metastasectomy. The underlying tumor biology is
considered to be the main cause for heterogeneity in the outcome of
patients with CRC lung metastases. Several prognostic
biomarkers have been proposed to define aggressive tumors
associated with fatal outcome [
2–4
]. During the last years,
the tumor microenvironment gained increasing attention in
the scientific community, especially in groups focusing on
metastatic CRC [
5–7
].
Immune escape is considered an emerging hallmark of
cancer [
8
]. Various subsets of lymphocytes can be found in
the tumor microenvironment, so called tumor-infiltrating
lymphocytes (TILs). They can launch pro-inflammatory
anti-tumor responses or mediate local immunosuppression.
The amount of TILs has a prognostic value in various
primary solid cancer types, including lung, renal, breast
and CRC [
9–14
]. Commonly detected lymphocyte subsets
with favorable prognostic impact are mature T-cells
[cluster of differentiation (CD)3?] and cytotoxic T-cells
(CD8?), memory-T-cells (CD45RO?), while immune
suppressive regulatory T-cells (FoxP3?) are associated
with impaired prognosis. Moreover, tertiary lymphoid
structures (TLSs), which are ectopic lymphoid aggregates
present in chronically inflamed tissue, can be found in the
tumor stroma. TLS are believed to promote and maintain
inflammation and anti-tumor response similar to secondary
lymphoid organs. The presence of TLS in the tumor
microenvironment is associated with favorable prognosis
especially in CRC [
15, 16
].
So far little is known about the local immune response
in CRC metastases, as previous studies focused on primary
tumor specimen. Metastasis initiating cells have already
successfully conquered immune escape during
intravasation, survival in the blood stream and extravasation
indicating that the composition of the immune
microenvironment might differ from the primary tumor
[
17, 18
]. Therefore, we aimed to investigate the local
inflammatory microenvironment in CRC PM specimen and
matched primary CRC specimen.
Materials and methods
Study population
From April 2009 to June 2014 57 patients with primary
CRC receiving complete/curative pulmonary
metastasectomy at the Medical University of Vienna were
retrospectively recruited from a prospective institutional
database. The study was approved by the Institutional
Ethics Committee (# 1035/2014) and conducted according
the Declaration of Helsinki. Resected pulmonary nodules
were verified as metastases from primary CRC by a
board certified pathologist. Additionally, samples of the
corresponding primary tumor were available in 31/57
(54.4 %) patients. The patients underwent post-surgical
tumor surveillance after pulmonary metastasectomy
including periodical computed tomography (CT) scans.
Immunohistochemistry
Immunohistochemical (IHC) staining was performed on
4 lm thick sections of formalin-fixed, paraffin-embedded
tissue samples using an automated staining platform
(Ventana Benchmark Ultra immunostainer, Ventana
Medical Systems, Inc., Tucson, USA).
Immunostaining was performed with anti-CD3 (clone
SP7, #RM9107-S1, Thermo Fisher Scientific, Cheshire,
UK), anti-CD8 (clone C8/144B, #M7103, Dako, Glostrup,
Denmark), anti-CD45RO (clone UCHL1, #M074201,
Dako, Glostrup, Denmark) and anti-FOXP3 (clone 206D,
#320116, BioLegend, San Diego, CA, USA) antibodies
using an autostainer (Benchmark Ultra, Ventana Medical
Systems, Tucson, USA) according to the manufacturer’s
instructions. In negative controls the primary antibody was
omitted. A mediastinal lymph node served as positive
control. The presence of lymphatic vessel invasion in PM
was known from a previous study [
19
].
Quantification and scoring of TILs and TLS
The density of CD3?, CD8?, CD45RO? and FoxP3?
TILs was evaluated in a semiquantitative manner on full
size sections as described previously [
20, 21
]. In summary,
scores reaching from 0 (absent infiltrate) to 4? (very dense
infiltrate) were assigned to the tissue samples. First, an
overall impression was rated at low magnification (9100).
Additionally, the spatial distribution of the immune
infiltrate was assessed in the tumor center and at the invasive
margin separately at higher magnification (9200–400). For
further analyses, the TILs densities were dichotomized.
The used cut-offs are provided in Supplementary Table 1.
Moreover, the sections were screened for the presence of
TLSs based on morphologic features (TLS; present vs. not
present). Follicular aggregates of lymphatic cells were
defined as TLS, whereas perivascular lymphatic aggregates
were excluded. The presence of CD3?, CD8?, CD45RO?
and FoxP3? cells in the TLS was assessed and, according
to TILs quantification, scores from 0 to 4 were assigned to
the samples. In general, negative and sparse infiltration (0
and 1?) were grouped in contrast to moderate, dense and
very dense infiltration (2?, 3? and 4?), which led to two
group sizes as equal as possible. As CD3 were
omnipresent, a higher cut-off had to be applied. Also for CD8 TILs,
a higher cut off was chosen as described previously by
others [
22, 23
]. The dichotomized variables were used for
further calculations. CD8/FoxP3 ratios were calculated
from dichotomized values. Samples with high CD8/low
FoxP3 were described as CD8/FoxP3-ratio high, samples
with high CD8/high FoxP3 or low CD8/low FoxP3 as equal
and low CD8/high FoxP3 were described as low. Two
observers (TS, ASB) blinded to the clinical data rated each
section independently using a multi-head microscope. If
the rating differed, the slide was re-discussed and a
consensus was found.
Statistical analysis
The disease-free interval (DFI) was defined as the time
between surgery for the primary tumor to pulmonary
metastasectomy in months. Time to recurrence represented
the time between pulmonary metastasectomy and evidence
of recurrence at any organ site. Overall survival (OS) was
defined as the period of time between pulmonary
metastasectomy and death of any cause. If patients had a history
of pulmonary metastasectomy before the inclusion period,
the previously resected metastases were assessed and the
outcome was calculated from the first pulmonary
metastasectomy. Nominal variables were compared using v2 test
or Fisher’s exact test (if expected frequency \5). Survival
curves were estimated using Kaplan–Meier plots and the
difference of the groups were compared using the log-rank
test. Statistically significant variables (P-values B0.05) in
the univariate analysis were added to a multivariate Cox
regression model. Statistics were performed using SPSS 23
(SPSS, Inc., Chicago, USA) and GraphPad Prism 6
(GraphPad Software, Inc., California, USA) software. All
performed tests were two-sided. P-values B0.05 were
considered statistically significant. Due to the hypothesis
generating approach of the study no correction for multiple
testing was used [
24
].
Results
Fifty-seven patients with histologically verified PMs from
primary CRC were included in this study. Macroscopically
and microscopically complete resection was achieved in all
patients. 33 (57.9 %) patients were male and 24 (42.1 %)
female. Median age at the time of pulmonary
metastasectomy was 64 years (range 33–83). The primary tumor site
was colon in 32 (56 %) patients and rectum in 25 (44 %)
patients. A detailed description of the patients’
characteristics is provided in Table 1.
Density and distribution of TILs and TLS in pulmonary metastases
Sufficient IHC quality was achieved in 55/57 (96.5 %),
57/57 (100 %), 54/57 (94.7 %) and 53/57 (93.0 %) of PM
specimen for CD3?, CD8?, CD45RO? and FoxP3? TIL
evaluation, respectively. CD3?, CD8?, CD45RO? and
FoxP3? TILs at variable density were evident in 55/55
(100 %), 55/57 (96.5 %), 50/54 (92.3 %) and 45/53
(84.9 %) of PM. A detailed description of the density and
spatial distribution of TILs is shown in Table 2.
Representative images of TILs are provided in Fig. 1. TLS were
present in 45/57 (78.9 %) PM specimen. If TLS could be
found, the density of CD3?, CD8?, CD45RO? and
FoxP3? T-cells in TLS was assessed (Supplementary
Table 2) and correlated with clinicopathological
characteristics (Supplementary Table 3).
The density of CD3?, CD8?, CD45RO? and FoxP3?
TILs was correlated with clinicopathological characteristics
of our patients. Intratumoral FoxP3? TILs were more often
present in patients with single PM (v2 P = 0.011). All (10/10
(100 %) patients with a DFI of more than 60 months had
high levels of CD45RO? TILs at the invasive margin,
compared to 24/36 (66.6 %) patients in the group DFI
\36 months (DFI [60 vs. \36 months; Fisher’s exact test
P = 0.044). FoxP3? TILs at the invasive margin were
associated with evidence for lymphatic vessel invasion (v2
test P = 0.050). High CD3? infiltration in the tumor center
was more often found in PM from colon cancer compared to
rectal cancer (v2 P = 0.040). Otherwise, no association of
TILs density with age, sex, tumor localization, UICC stage of
the primary tumor, previous liver metastasis or
chemotherapy before metastasectomy was evident (Table 3).
Correlation of TILs and TLS in pulmonary metastases and corresponding primary CRC
A detailed description of CD3?, CD8?, CD45RO? and
FoxP3? TILs density in the tumor center and at the invasive
margin of the corresponding primary tumors is provided in
Supplementary Table 4. We found no significant correlation
between the TIL density in the primary tumor and
corresponding lung metastases (Supplementary Table 5). In
general, PM had higher densities of CD3?, CD8?,
CD45RO? TILs, whereas the FoxP3 TIL were comparable
(Supplementary Fig. 1). Significantly less primary CRC
were rated as TLS positive compared to the paired PM
samples [2/28 (7.1 %) vs. 22/28 (78.6 %); McNemar test
P \ 0.001].
Impact of TILs and TLS in PM on outcome parameters
The presence of FoxP3? TILs at the invasive margin was
significantly associated with a decreased OS (35 vs.
65 months; HR 2.40; 95 % CI 1.11–6.96; log-rank test
P = 0.031) (Fig. 2). Moreover, dense CD8? infiltrates at
the invasive margin were associated with improved OS
Total study cohort
(n = 57)
n
(median survival not reached vs. 39 months; HR 0.00;
95 % CI 0.09–1.04; log-rank test P = 0.064).
6/29 (20.7 %) patients had a high CD8/FoxP3-ratio,
CD8/FoxP3 was equal in 10/29 (34.5 %) patients and a low
ratio was found in 13/29 (44.8 %) patients. The CD8/
FoxP3-ratio had significant impact on OS prognosis after
pulmonary metastasectomy (log-rank test P = 0.021 and
0.042 for low vs. equal and low vs. high, respectively).
Furthermore, patients with a high CD8/FoxP3-ratio at the
invasive margin had a prolonged recurrence-free survival
after pulmonary metastasectomy compared to patients with
low CD8/FOXP3 ratio (26 vs. 15 months; HR 0.32; 95 %
CI 0.12–1.03; log-rank test P = 0.080).
The presence of TLSs in PM alone was neither
associated with recurrence-free survival (log-rank test
P = 0.141) nor with OS (P = 0.813). However, when
assessing the T cell subsets of TLS, we found a significant
association of high levels of CD8? cells in TLS and
improved OS (median survival not reached vs. 35 months;
HR 0.30; 95 % CI 0.14–0.79; log-rank test P = 0.016).
Similarly to TILs, a strong prognostic effect was also found
for the CD8/FOXP3-ratio in TLS (high vs. low HR 6.99;
95 % CI 1.28–20.48; log-rank test P = 0.027). Outcome
analyses of CD3?, CD8?, CD45RO? and FoxP3? cells
in TLS are summarized in Table 4 and Fig. 3. Adding
CD8? in TLS and FoxP3? at the invasive margin into a
multivariate Cox regression model, only CD8? cells in
TLS remained a significant prognosticator for OS after
pulmonary metastasectomy (P = 0.020; HR 0.29; 95 % CI
0.07–0.79).
%
CD45RO? TILs
Tumor center
Low
High
The aim of this study was to evaluate the role of TILs and
TLSs in PM assessing a cohort of patients with CRC lung
metastases. CD3? TILs were found in every resected
pulmonary metastatic specimen, highlighting the pivotal
role of the adaptive immune system in local tumor
microenvironment. We could show that tumor infiltrating
CD8? and FoxP3 positive cells were associated with
disease free survival after pulmonary metastasectomy and OS.
CD8? T cells represent a subpopulation of T cells, also
known as cytotoxic T cells. They play an important role in
the defense against viruses but also cancer cells. Upon
activation they release cytotoxins (e.g., perforin,
granzymes, granulysin) into infected or tumorous somatic cells,
which eventually leads to the induction of apoptosis.
Tumor infiltrating CD8? cells can induce a potent
tumorlytic response, which has been shown for various
malignancies [
25
].
FoxP3? cells are known as regulatory T-cells (Tregs).
The have the ability to suppress effector T-cell function
both in a paracrine and cell–cell-contact dependent manner
[
26
]. Tregs are important for the maintenance of
immunological tolerance, however, can also dampen
antitumor response of the immune system. An expansion of the
Treg pool experimentally leads to enhanced vulnerability
of carcinogens and worse outcome [
27, 28
].
The role of TILs has been extensively studied in primary
CRC. The inflammatory infiltrate was shown to correlate
with the T-stage of primary CRC and even allowed a more
precise prognosis on patients’ outcome compared to the
UICC–TNM staging alone [
10
]. In the subgroup of rectal
cancer patients, the prognostic value of the immune
infiltrate (CD3 and CD8) was confirmed and additionally found
to be a predictive marker for the response to preoperative
chemo-radiotherapy [
9
]. Based on this data, an
international consortium was founded to standardize and
implement an adapted staging system taking the immune
infiltrate (‘‘Immunoscore’’) into account [
29
].
In contrast to the evidence of the role of TILs in primary
CRC, sparse data exists on TILs in lung metastases. To the
best of our knowledge, by now only two studies have
0.393
0.172
0.547
examined TILs in CRC lung metastases. Remark et al.
showed in a retrospective cohort with CRC PM that a high
density of CD8? TILs conferred an improved OS
(P = 0.039 in univariate analysis). Interestingly, CD8?
TILs at the tumor center and at the invasive margin had a
similar prognostic impact [
30
]. In another study conducted
in a Korean patient cohort with UICC stage IV colon
cancer, including 21 patients with PM and 58 with liver
metastases, a high CD8? TILs density in the primary
tumor, but not in the metastatic tissue (liver and lung
together) had a beneficial impact on OS (log-rank test
P = 0.017 and 0.232, respectively). The strongest positive
prognostic impact was found for high CD45RO? TILs in
the primary and metastatic tissue (log-rank test P = 0.009
and 0.027, respectively). The prognostic impact of
CD45RO? TILs in the primary tumor remained significant
in a multivariate model (RR 0.108; 95 % CI 0.021–0.546;
P = 0.007). Interestingly, a high density of FoxP3? TILs
at the metastatic site was also associated with a favorable
prognosis (log rank test P = 0.050) in this study [
31
]. This
is in contrast to most of the published evidence on
immunosurveillance in CRC and this finding could not be
confirmed in our patient cohort [
32, 33
]. This discrepancy
might be explained by the mixture of liver (73 %) and lung
metastases (27 %), which were not separated in the
outcome analysis [31]. Another explanation might be the
spatial distribution of FoxP3 cells. Salama et al. reported a
decreased survival of stage II CRC patients with low
content of FoxP3? T-cells within the tumor (HR 0.65;
95 % CI 0.48–0.89; P = 0.007), whereas the opposite was
found when assessing adjacent colonic tissue (HR 1.42;
95 % CI 1.05–1.92; P = 0.023) [
11
]. The authors
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DFI disease-free survival to first pulmonary metastasis, IM invasive margin, NR 50 % recurrence/survival not reached, TC tumor center, TILs
tumor-infiltrating lymphocytes, TLS tertiary lymphoid structure
speculated that FoxP3? T-regulatory cells might lose their
ability to suppress antitumor immunity when they are
found within the tumor tissue. Our results suggest differing
impact of FoxP3? TILs depending on their localization.
Dense infiltration of FoxP3? TILs in the infiltration zone
was associated with impaired survival, whereas infiltration
of FoxP3? TILs in the tumor center did not alter survival.
Moreover, high levels of FoxP3? TILs at the invasive
margin were significantly more common in patients with
evidence for lymphatic invasion, which is believed to be a
key-determinant of the outcome after pulmonary
metastasectomy [
19, 31
]. Similar to two recent publications, which
described the ratio between CD8? and FoxP3? TILs as
prognostic factor superior to CD8? and FoxP3? TILs
alone, we found the CD8/FoxP3- ratio to have a strong
impact on OS in our patient cohort [
34, 35
]. In general, our
observations in PMs imply differential roles of TILs
dependent on their localization within the metastatic tissue.
This has to be taken in consideration when assessing TILs
in histological specimen and when defining cut-offs for
statistical evaluation.
The present work is, to the best of our knowledge, the
first analysis of presence and composition of TLS in CRC
lung metastases. We could demonstrate that high levels of
CD8? cells as well as a high CD8/FoxP3-ratio in TLS
correlated positively with patients’ OS after pulmonary
metastasectomy (P = 0.016 and 0.027, respectively).
Similar to our findings on TILs at the invasive margin, the
balance of CD8? and FoxP3? in TLS seems to be a
crucial factor for the survival of CRC patients with
dissemination to the lung. Di Caro et al. found that TLS were
present in 78.6 % of stages II and III primary CRC. The
presence of TLS was a positive prognosticator only in stage
II CRC (log rank P = 0.02), whereas the prognostic impact
was lost in an advanced tumor stage (UICC stage III) [
16
].
Also in our cohort of stage IV CRC patients, the presence
of TLS alone had no impact on recurrence-free and OS.
Salama et al. evaluated the distribution of FoxP3? cells in
TLS in stage II colon cancer and described the high density
of FoxP3? cells in TLS as negative prognostic factor of
OS (HR 4.22; 95 % CI 1.49–11.91; P = 0.007 in
multivariate analysis) [
36
]. In our cohort, patients with
high number of FoxP3? cells in TLS evidenced a
decreased recurrence-free and OS, without reaching the
level of significance. Only 7.1 % of the primary CRC
samples were rated as TLS positive in our cohort. This
might indicate an insufficient immune response at the
primary tumor site in patients subsequently developing
metastatic disease. However, caution is warranted when
interpreting this finding due to the small sample size.
Interestingly, the density of the immune infiltrate in
primary tumors did not correlate with the infiltrate in PM.
Considering the heterogeneity of tumors, the metastatic
spreading of subpopulations of tumor cells of the primary
cancer to distant organs might explain the different
immuno-phenotype observed in our cohort [
37
]. In
general, lung metastases appear to consist of more
immunogenic tumor cells compared to the primary tumor
site, since the density of CD3?, CD8? and CD45? TIL
increased during progression of disease. Another possible
explanation for this observation might be the unique
immunological state of the lung. It is constantly exposed
to environmental pathogens and therefor rich in residing
immune cells.
Our findings suggest that patients with CRC lung
metastases is a heterogeneous group regarding the tumor
biology. This might have implications on the management
of these patients. Histological evidence for an aggressive
tumor biology might have consequences regarding the
offered adjuvant chemotherapy, tumor surveillance
strategies and a possible future re-metastasectomy.
There are several limitations to this study. First, no
uniform protocol for pseudo-neoadjuvant and
pseudo-adjuvant chemotherapeutic regimens was applied. According
to the preferences of the referring oncologist, different
schemes of chemotherapy were administered to the
patients. As chemotherapeutic agents might contribute to
the immune-editing of tumors, this is a possible confounder
of this study. However we could not find a significant
association between administration of chemotherapy before
metastasectomy and the immune infiltrate in PMs.
Moreover, the history of chemotherapy before metastasectomy
did not affect the outcome after lung metastasectomy.
Second, patients eligible for surgery are a subgroup and do
not represent the whole population of patients with lung
metastases from CRC. Thus, our findings might not be
extrapolated to all patients with CRC PMs due to this
selection bias. Last, although this is currently the biggest
cohort of patients with CRC PM in which the CD3?,
CD8?, CD45RO? and FoxP3? TILs and TLS was
assessed, the study might still be underpowered. A
multiinstitutional study with a considerable sample size will be
necessary to further clarify the prognostic value of TILs
and TLS in CRC lung metastases.
In summary, this is the first structured analysis of
CD3?, CD8?, CD45RO? and FoxP3? TILs and TLS in
the setting of curative pulmonary metastasectomy,
including more than 50 patients with CRC lung metastases. Our
results suggest that especially the balance of effector
CD8? and regulatory FoxP3? TILs play a crucial role
during immune-editing of metastatic CRC and thus,
predicting outcome of patients after pulmonary
metastasectomy with curative intent.
Acknowledgments This study was supported by a Research Grant
(#15880) provided by the Austrian Federal Bank (OeNB), by a Grant
provided by Initiative Krebsforschung (Project Title
‘‘Tumorimmunologie von Hirnmetastasen’’) and by the Christian Doppler
Laboratory for Cardiac and Thoracic Diagnosis and Regeneration.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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