The Prognostic Significance and Relationship with Body Composition of CCR7-Positive Cells in Colorectal Cancer
George Malietzis MBBS MSc MRCS1,2,,Gui Han Lee MBBS MRCS1,2,3, David Bernardo PhD1, Alexandra I F Blakemore PhD4, Stella C Knight PhD1, Morgan MoorghenMD FRCPath3, Hafid O Al-Hassi PhD1, John T Jenkins MD FRCS2
- Antigen Presentation Research Group, Imperial College London, North West London Hospitals Campus, Watford Road, Harrow HA1 3UJ, UK
- Department of Surgery St Marks Hospital, Watford Road, Harrow, Middlesex, HA1 3UJ, UK
- Department of Histopathology St Marks Hospital, Watford Road, Harrow, Middlesex, HA1 3UJ, UK
- 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:
Telephone: +44 20 8869 3494
Fax: +44 20 8869 3532
Short Title: CCR7andColorectal Cancer
Funding sources: noneDisclosures: 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-embeddedtissue specimens was carried outin 116patients with non-metastatic CRC. CCR7expression was determined by immunohistochemistry. Analysis ofcomputer tomography scans was used to calculate BC parameters. Survival analyses and multivariate regression models were used.
Results: High CCR7+ cell density within the tumorstroma and at the margin was significantly associated withincreased 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)(p0.001). 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.
Virchowfirst 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 tumoris associated to these changes. [6]Body composition defined the proportions of fat, muscle and bone of an individual. [7] Muscledepletion is characterized by reduction in muscle size (myopenia) and anincreased infiltration by inter- and intramuscular fat, described asmyosteatosis.[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, andmature dendritic cells, and is considered to play an importantrole 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 involvedin the lymphatic spread of solid tumors.[13]Evidence suggests that assessment of the CCR7expression on CRCs specimensmight 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 investigateits 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 computerisedtomography(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 ormetastatic 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,TNMstage (UICC 5 version), the presence of vascular invasion and histopathologicalgrade of differentiation.
Tissue Samples
Colorectal cancer paraffin embedded tissue blockswere obtained from all the patients meeting the selection criteria.
Immunohistochemical Analysis
Preparation of sections from paraffin blocks was performed by standard methods. Immunohistochemicalanalysis of CCR7 was performed using a mouse monoclonalantibody against human CCR7 (CCR7 MAb (Clone 150503) Cat# MAB197) according to standardtechniques for a Ventana BenchmarkXTAutostainer (Ventana Medical Systems). Antigen retrieval was carried out using Cell ConditioningSolution (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.Thecell density defined as the percentage of the area containing CCR7+ cells (summedarea with CCR7+cells / total measured area x 100) wascalculated 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 lowgrade (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 wasnormalised 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 outcomeswere 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, 27had 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 of15.85 % (Inter Quartile Range (IQR)10.02-21.83 %) inthe tumor stroma, and 7.17 % (IQR 3.90-12.37 %) at the tumormargin. CCR7+ cell density ofthe two areas correlated positively (Spearman r = 0.77;p0.001). The median CCR7+ cell density for the tumour cells was 16.78 % (IQR 7.28-22.76). We divided the cases into highand 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 thetumor center and the margin was significantly associatedwith olderage, 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 lymphovascularinvasion. There was no significant association between CCR7+ cell density either at the margin or within an intra-tumoral locationwith 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 metastasisand 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 duringa median 40-month follow-up (IQR15-50 months).Kaplan–Meier analysis demonstrated that high CCR7+ cell density at the tumormargin wassignificantly associated with shorter disease-free andoverall survival (log-rank test, p=0.031 and p=0.022; respectively). Figure 2 demonstrates the Kaplan-Meier graphs of CRCoverall survival (OS) and disease free survival (DFS) after resection for CRC according to CCR7+ cell density at the tumor marginand 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 tumormargin 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 inthe tumormargin or in the tumour cells was not anindependent prognostic factor for DFSorOS in this study. Lymph node metastasis and grade of differentiation were identified as beingindependently 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, lymphnode metastasis, high grade of inflammatory response (KM score), and myosteatosisthat are adverse prognostic factors inCRC. Moreover, high CCR7+cell density in the tumor margin was significantly associated with shorterDFS and OS. Our findingssuggest that tumor-infiltrating CCR7+ cells areassociated with a more aggressive cancer.
The mechanisms by which LIR affects prognosis in patients with CRC are not clear. We foundan 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 noveland 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 ofCCR7.[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-tumorcells 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, Schimanskiet 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. Correaleet 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-metastaticCRC,multivariate analyses demonstratedthat 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 withearly 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 peritumouralinflammatory 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 onthe 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 ofdetermining the characteristics of tumor infiltrating inflammatory cells, rather than only the density or count of inflammatory cells determined by KM score.