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: s.knight@imperial.ac.uk

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

(b,d).