The Prognostic Significance and Relationship with Body Composition of CCR7-Positive Cells in Colorectal Cancer
George Malietzis MBBS MSc MRCS 1,2,, Gui Han Lee MBBS MRCS 1,2,3, David Bernardo PhD 1, Alexandra I F Blakemore PhD 4, Stella C Knight PhD 1, Morgan Moorghen MD FRCPath3, Hafid O Al-Hassi PhD 1, John T Jenkins MD FRCS 2
1. Antigen Presentation Research Group, Imperial College
London, North West London Hospitals Campus, Watford Road,
Harrow HA1 3UJ, UK
2. Department of Surgery St Marks Hospital, Watford Road,
Harrow, Middlesex, HA1 3UJ, UK
3. Department of Histopathology St Marks Hospital, Watford
Road, Harrow, Middlesex, HA1 3UJ, UK
4. Section of Investigative Medicine, Division of Diabetes,
Endocrinology, and Metabolism, Faculty of Medicine, Imperial
College, London W12 0NN, UK
Corresponding Author:
Professor Stella C Knight
Antigen Presentation Research Group, Imperial College London,
North West London Hospitals Campus, Watford Road, Harrow, HA1
3UJ, United Kingdom
Email: [email protected]
Telephone: +44 20 8869 3494
Fax: +44 20 8869 3532
Short Title: CCR7 and Colorectal Cancer
Funding sources: none Disclosures: none
Synopsis: High CCR7+ cell density in colorectal cancer was
associated with higher stage of disease, myosteatosis and overall
worse survival. Our results suggest that a specific immune
microenvironment may be associated with altered host’s body
composition and disease outcomes.
Abstract
Background and Objectives: The host local immune response (LIR)
to cancer is a determinant of cancer outcome. Regulation of this
local response is largely achieved through chemokine synthesis
from the tumor microenvironment such as C-Chemokine-Receptor-7
(CCR7). We examined the LIR measured as CCR7 expression, in
colorectal cancers (CRC) and explored relationships with body
composition (BC) and survival.
Methods: A study of paraffin-embedded tissue specimens was
carried out in 116 patients with non-metastatic CRC. CCR7
expression was determined by immunohistochemistry. Analysis of
computer tomography scans was used to calculate BC parameters.
Survival analyses and multivariate regression models were used.
Results: High CCR7+ cell density within the tumor stroma and at the
margin was significantly associated with increased age, the
presence of lymphovascular invasion, higher tumor stage, lymph
node metastasis, high Klintrup-Makinen immune score and
myosteatosis. High CCR7+ cell density in the tumor margin was
significantly associated with shorter disease-free (DFS) and overall
survival (OS)(p<0.001). This was also significantly associated with
shorter survival in multivariate analysis (HR=8.87; 95%CI(2.51-
31.3); p<0.01 for OS and HR=4.72; 95%CI(1.24-12.9); p=0.02 for
DFS).
Conclusions: Our results suggest that a specific immune
microenvironment may be associated with altered host’s BC and
tumor behavior, and that CCR7 may serve as a novel prognostic
biomarker.
Keywords: colorectal cancer, immunology, body composition,
CCR7, chemokines, outcomes
Introduction
Colorectal cancer (CRC) is the fourth most common cause of cancer
death and the third most common cancer worldwide. Colorectal
cancers are classified according to the TNM classification system.
Treatment planning is based on combinations of the tumor local
invasion depth (T-stage), the presence of positive lymph node (N-
stage) and distant metastasis (M-stage).[1] Although the TNM
staging system provides useful prognostic information, an individual
patient outcome from therapy cannot be accurately predicted.
Therefore, there is a need for additional prognostic markers to
complement the TNM system.
Virchow first described the link between cancer and inflammation,
suggesting that the “lymphoreticular infiltrate” at sites of chronic
inflammation reflected the origin of cancer.[2] The local tumor
microenvironment plays an important role in carcinogenesis
including, cell growth, invasion and metastasis and these effects are
mediated via host-derived stromal cells and cytokines. Jass first,
in 1986 proposed that infiltration of immune cells can act as an
independent prognostic factor in CRC, and since then the local
inflammatory response (LIR) has been accepted as a major factor in
the pathogenesis of cancer. [3] The LIR is associated with changes
in the type, density, and location of immune cells in cancer tumors
and also has been linked with weight and lean muscle loss. [4],[5]
Emerging data support the link between systemic inflammatory
response and body composition alterations but limited information
exists on how the LIR to the tumor is associated to these changes.
[6] Body composition defined the proportions of fat, muscle and
bone of an individual. [7] Muscle depletion is characterized by
reduction in muscle size (myopenia) and an increased infiltration by
inter- and intramuscular fat, described as myosteatosis.[8] Visceral
obesity is defined as the excess of intra-abdominal adipose tissue
accumulation.[9] These conditions are recognized as poor
prognostic indicators in patients with cancer. [10]
Originally, chemokines and their receptors were reported to mediate
different pro- and anti-inflammatory responses.[11] LIR depends on
the ability of immune cells to actively migrate in and out of tissue,
and chemokines are established regulators of immune cell migration
and survival. Two essential chemokines involved in cell movement
during homeostasis are CC-chemokine ligand 19 (CCL-19) and 21
(CCL-21), that are ligands for the CC-chemokine receptor 7 (CCR-7).
CCR7 is expressed on naıve T cells, memory T cells, B cells, and
mature dendritic cells, and is considered to play an important role in
lymphocyte cell trafficking and homing to lymph nodes. [12] In
cancer, CCR7 expression on immune cells regulates homing of
lymphocytes into secondary lymphoid organs and may also be
involved in the lymphatic spread of solid tumors. [13] Evidence
suggests that assessment of the CCR7 expression on CRCs
specimens might improve prediction not only of the survival
outcome but also of lymph node spread. [14]
In the present study, we aimed to determine the expression of CCR7
on tumour infiltrating cells in primary CRC and investigate its impact
on disease progression and survival. We also aimed to correlate the
expression of CCR7 with the patients’ clinical and pathological
parameters (including their body composition) derived from
computerised tomography (CT) analysis, [15] and to explore the
relationship between body composition and tumor immunology in
CRC.
Materials and Methods
Study population
A total of 242 consecutive patients with primary CRC who
underwent elective resection at St Mark’s Hospital between January
2009 and December 2011 were identified from a prospective
database. Patients with recurrent or metastatic disease confirmed
preoperatively or at surgery, emergency cases, those receiving
neoadjuvant chemotherapy and/or radiotherapy, and those with a
non-available pre-operative CT were excluded. All recorded clinical
and pathological data were revalidated from medical and pathology
records. Data collected prospectively during the perioperative
period included age, sex, Body Mass Index (BMI), histological
grading, TNM stage (UICC 5 version), the presence of vascular
invasion and histopathological grade of differentiation.
Tissue Samples
Colorectal cancer paraffin embedded tissue blocks were obtained
from all the patients meeting the selection criteria.
Immunohistochemical Analysis
Preparation of sections from paraffin blocks was performed by
standard methods. Immunohistochemical analysis of CCR7 was
performed using a mouse monoclonal antibody against human CCR7
(CCR7 MAb (Clone 150503) Cat# MAB197) according to standard
techniques for a Ventana Benchmark XT Autostainer (Ventana
Medical Systems). Antigen retrieval was carried out using Cell
Conditioning Solution (CC1-Tris-based EDTA buffer, pH 8.0; Ventana
Medical Systems).
Image Analysis
Images of immunostained slides were digitized at 40X magnification
using the Leica SCN400F. For digital quantification, image analysis
software (Tissue Studio v.3.5; Definiens AG, Munich, Germany) was
used to distinguish the CCR7+ cells. We focused on three main
regions of interest (tumor margin, tumor stroma and tumour). Two
independent assessors (GM and GHL) graphically mapped these
regions using the image software. Cells were considered to either
positive (+) or negative (-) according to presence of clearly defined
positively stained cytoplasm in a granular distribution. Faint ill-
defined staining was considered to represent an artifact and
considered negative. The image analysis software was calibrated
accordingly. The cell density defined as the percentage of the area
containing CCR7+ cells (summed area with CCR7+ cells / total
measured area x 100) was calculated for each slide.
Immune score
In an attempt to directly relate CCR7 expression with LIR, a
previously proposed method for assessing the LIR in CRC, the
Klitrup-Makinen (KM) grade was applied.[16] Briefly, using the
corresponding H&E-stained sections of the study population,
inflammatory cell infiltration at the invasive margin was graded
using a four-point scale and subsequently classified as low grade (no
increase or mild/patchy increase in inflammatory cells) or high
grade (prominent inflammatory reaction forming a band at the
invasive margin, or florid cup-like infiltrate at the invasive edge with
destruction of cancer cell islands), by two independent assessors
(GM and GHL). Discrepancies were resolved by an independent third
reviewer (MM).
Body Composition Analysis
Images were retrieved from digital storage in the Picture Archiving
and Communication System [PACS]. CT image analysis Slice-O-Matic
V4.3 software (Tomovision, Montreal, Canada) was performed as
described previously. [17] Briefly, total skeletal muscle and visceral
adipose tissue (VAT) surface area (cm2) were evaluated on a single
image at the third lumbar vertebrae (L3) using Hounsfield unit (HU)
thresholds of -29 to 150 for skeletal muscle, -50 to 150 for visceral
adipose tissue and -190 to -30 for subcutaneous adipose tissues.
The sum of skeletal cross-sectional muscle areas was normalised for
stature (m2) and reported as LSMI (cm2m-2). Mean Muscle
Attenuation [MA] (HU) was reported for the whole muscle area at
the third lumbar vertebra level. Reduced L3 skeletal muscle index
(myopenia) and low MA (myosteatosis) were defined using
predefined sex-specific skeletal muscle index cut-points. [18]
Increased visceral adipose tissue area (visceral obesity) was also
described by using gender-specific and pathologically relevant cut-
off values. [9]
Statistical Analysis
The relationship between CCR7 expression and other
clinicopathological parameters was assessed using nonparametric
statistics. Clinical outcomes were assessed using the Kaplan-Meier
survival curves, and the groups were compared using the log-rank
test. Stepwise multivariate Cox proportion analysis was performed.
The level of significance permitting multivariate analysis inclusion
and the statistical significance for all other tests used was set at P <
0.05. All analyses were performed using the statistical software,
Statistical Package for the Social Sciences, version 20.0 (SPSS, Inc,
Chicago, IL).
Results
Patient Selection
Of 242 consecutive patients undergoing surgical resection, 42 cases
had a preoperative CT scan stored in a paper film form and,
therefore, unsuitable for analysis, 27 had had emergency surgery,
26 had recurrent or metastatic disease at the time of surgery, 8
received neo-adjuvant treatment, and for 17 the CT analysis was not
possible due to poor image acquisition quality. Exclusion of these
patients resulted in a sample size of 118 patients who had
undergone elective resection for CRC.
Distribution of CCR7+ cells in CRC
Staining was achieved in all 118 specimens with the majority
showing homogeneous staining but, as expected, different
intensities were frequently observed. Staining expression of CCR7
was observed mainly at the tumour margin, and stroma but also in
the primary tumour. Image software analysis from all the specimens
revealed a median tumor infiltrating CCR7+ cell density of 15.85 %
(Inter Quartile Range (IQR) 10.02-21.83 %) in the tumor stroma, and
7.17 % (IQR 3.90-12.37 %) at the tumor margin. CCR7+ cell density
of the two areas correlated positively (Spearman r = 0.77;
p<0.001). The median CCR7+ cell density for the tumour cells was
16.78 % (IQR 7.28-22.76). We divided the cases into high and low
CCR7+ groups according to the median value of CCR7 + cell
density. Figure 1 demonstrates the distribution of CCR7+ cells in
CRC.
CCR7+ cells and clinical and pathological parameters
High CCR7+ cell density at both the tumor center and the margin
was significantly associated with older age, higher tumor stage,
lymph node metastasis and the presence of myosteatosis. High
CCR7+ cell density at the tumor margin was also significantly
associated with female sex and the presence of lymphovascular
invasion. There was no significant association between CCR7+ cell
density either at the margin or within an intra-tumoral location with
BMI, site of tumor, grade of differentiation, myopenia or visceral
adiposity. A high KM grade was identified in 38 % of the cases
studies and this was significantly associated with the CCR7+ cell
density at the tumour margin but not in the stroma. High density of
CCR7+ tumour cells was significantly associated with higher tumor
stage, lymph node metastasis and the presence of lymphovascular
invasion. Table 1 demonstrates the correlation between tumor-
infiltrating CCR7+ cell density and clinicopathological factors in
patients with CRC who had been treated surgically.
CCR7+ cells and Clinical Outcome of Colorectal Cancer
Among the 118 patients, there were 13 recurrences and 18 deaths
during a median 40-month follow-up (IQR 15-50 months). Kaplan–
Meier analysis demonstrated that high CCR7+ cell density at the
tumor margin was significantly associated with shorter disease-free
and overall survival (log-rank test, p=0.031 and p=0.022;
respectively). Figure 2 demonstrates the Kaplan-Meier graphs of
CRC overall survival (OS) and disease free survival (DFS) after
resection for CRC according to CCR7+ cell density at the tumor
margin and the stroma.
To determine the independent prognostic significance of CCR7+ cell
density on DFS and OS, multivariate analysis using a Cox
proportional hazards model was performed. High CCR7+ cell density
at the tumor margin was significantly associated with shorter DFS
and OS in multivariate regression analysis (HR=8.87; 95%CI (2.51-
31.3); p<0.01 for OS and HR=4.72; 95%CI (1.24-12.9) p=0.02 for
DFS) as outlined in Table 2. However a high CCR7+ cell density in
the tumor margin or in the tumour cells was not an independent
prognostic factor for DFS or OS in this study. Lymph node
metastasis and grade of differentiation were identified as being
independently prognostic factors for OS and grade of differentiation
was also an independent prognostic factor for DFS.
Discussion
We found that a high density of tumor-infiltrating CCR7+ cells was
significantly associated with age, histological invasion, higher tumor
stage, lymph node metastasis, high grade of inflammatory response
(KM score), and myosteatosis that are adverse prognostic factors in
CRC. Moreover, high CCR7+ cell density in the tumor margin was
significantly associated with shorter DFS and OS. Our findings
suggest that tumor-infiltrating CCR7+ cells are associated with a
more aggressive cancer.
The mechanisms by which LIR affects prognosis in patients with CRC
are not clear. We found an association between infiltration of CCR7+
cells, at the tumor margin and within the tumor stroma, with some
of the clinicopathological variables examined. In particular, high
CCR7 density in the tumor margin and stroma were directly
correlated with adverse prognostic factors such as increased age,
advanced T and N stage and the presence of myosteatosis. These
findings may therefore suggest a model whereby the stimulus for
the local immune cell response is not only induced by the tumor but
also influenced by host-related factors. Recent work from our group
has demonstrated an association between myosteatosis and the
presence of an altered systemic inflammatory response in patients
treated for CRC. [6] We have now identified that myosteatosis is
also related to an adverse local inflammatory response as measured
by a high CCR7 density. To our knowledge these findings are novel
and may support the hypothesis that host LIR may influence the
development and persistence of myosteatosis.
Retrospective studies on various cancers have shown that tumor
cells express CCR7, including breast, [19] melanoma, [20]
oesophageal, [21] lung, [22] head and neck [23] and CRC.[24]
Ongoing oncogenic mutagenesis within the tumor can lead to
increased expression of chemokine receptors including CCR7, but
also tumor-derived factors such as VEGF and PGE2 may contribute
to this over-expression.[25-27] Therefore, increased expression of
CCR7 on immune cells infiltrating the tumor mass can be attributed
to this tumor behavior. Increased on-going chemokine production by
the tumor will attract immune cells and up-regulate the expression
of CCR7. [28] In our study, we have specifically focused on the
critical interface between tumor and the stroma and the margin
excluding tumor tissue itself. Therefore, CCR7+ cells from our study
will mainly consist of immune cells known to express CCR7, such as
T cells, antigen presenting cells and stroma cells. This is further
supported by the observation that high expression of CCR7 at the
tumour margin was strongly associated with high grade of
inflammatory response measured with the KM score.
The impact of CCR7+ non-tumor cells on CRC outcomes has been
recorded previously, but the results were controversial: Gunther et
al. studied the expression of CCR7 on paraffin-embedded tumor
specimens of 99 all stages CRC patients and concluded that
increased CCR7 expression at the invasion margin was associated
with worse OS. [24] Similarly, Schimanski et al. studied the
expression of CCR7 and another chemokine receptor, CXCR4, on
tumor specimens of 96 CRC patients of all stages. [29] However,
only increased CXCR4 expression was associated with poorer
outcome, not CCR7 expression. Correale et al. studied the
expression of CCR7 on tumor-infiltrating T cells in 76 patients with
metastatic CRC. The results demonstrated that high expression of
CCR7 positive tumor infiltrating lymphocyte, specifically CD8+
CCR7+ cells, was predictive of good outcome in patients with
advanced CRC. [30] Previous studies have shown a beneficial role of
infiltrating CD8+ cells in outcome in CRC. [31],[32],[33] Therefore,
expression of CCR7 on CD8+ cells in CRC could correlate with
improved outcome, especially in advanced CRC. In the present
study, on a population of 118 patients with non-metastatic CRC,
multivariate analyses demonstrated that high CCR7+ cell density at
the tumor margin is significantly associated with shorter DFS and
OS. Our results suggest that CCR7+ cell density at the tumor margin
may be a novel prognostic biomarker to predict outcomes in
patients with early CRC.
Our analyses showed that in the tumor periphery, high CCR7+ cell
density was associated with high KM score. A recent study reported
that high KM score correlated with markers of infiltrated
peritumoural inflammatory cells (CD3, CD8, CD68 and FoxP3 cells),
but no association was identified with dendritic cell density
determined using CD1a+ cells. [34] Therefore, it is possible to infer
that CCR7 positivity in our study was mainly due to expression on
the inflammatory cells described above. Our results also suggest
that despite increased LIR in the tumor peripheries (demonstrated
by high KM score), high expression of CCR7 in these immune cells
may have an impact and key role for the development of an efficient
immune response. This highlights the importance of determining the
characteristics of tumor infiltrating inflammatory cells, rather than
only the density or count of inflammatory cells determined by KM
score.
This study has a number of limitations. The identification and
classification of specific cell types expressing CCR7 was not
performed. However, the main aim of this work was to investigate
the overall stromal expression of CCR7 in the tumor margin and the
stroma of the colorectal tissues and whether this expression is an
indicator of undesirable prognosis in patients with CRC. Results were
encouraging and pave the way to assess the prognostic value of the
expression of CCR7 on particular cell types, which will be the subject
of future work. Although CCR7 expression was observed on the
tumour cells we focused only on the tumor margin and stroma. The
primary reason for this approach is that the tumor stroma and
margin represent a vital compartment of the tumor
microenvironment that reflects LIR, affects tumor progression and
metastasis [35] and also because the expression of CCR7 on tumor
cells has been previously reported.
In summary, our data give additional support to the prognostic
significance of the LIR in CRC. Moreover, our results suggest that
CCR7 positive cell density at the tumor margin may be a novel
prognostic biomarker to predict outcomes in patients with CRC.
Acknowledgements
The authors thank Matt Ellis for recommendations and assistance
and R. Baldwin for retrieving and preparing for analysis the CT
images. HOA was supported by a grant from the Association of
International Cancer Research (AICR) Scotland, Grant number
120234.
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Figure Legends
Figure 1 CCR 7 cell immunohistochemistry shows high (A,C) and low
(B,D)
infiltration of CCR7+ cells in the stroma (A,B) and the tumour
periphery
(C,D).
Figure 2 Kaplan-Meier graphs of colorectal cancer overall survival
(OS) (a,b)
and disease free survival (DFS)(c, d) after resection for CRC
according
to CCR7+ cell density at the tumour periphery (a,c) and stroma