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The Prognostic Significance and Relationship with Body Composition of CCR7Positive 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 genderspecific 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 tumorinfiltrating 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 overexpression.[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 upregulate 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 (b,d).