Prognostic Value of The Tumour-Infiltrating Dendritic Cells in Colorectal Cancer: A Systematic Review
George Malietzis1,2,Gui H Lee1,2, John T Jenkins2, David Bernardo1, MorganMoorghen3, Stella CKnight1, Hafid O Al-Hassi1
- Antigen Presentation Research Group, Imperial College London, North West London Hospitals Campus, Watford Road, Harrow HA1 3UJ, United Kingdom
- Department of Surgery St Marks Hospital, Watford Road, Harrow, Middlesex, HA1 3UJ, United Kingdom
- Department of Histopathology St Marks Hospital, Watford Road, Harrow, Middlesex, HA1 3UJ, United Kingdom
Corresponding Author
Professor Stella C. Knight
Antigen Presentation Research Group Imperial College London North West London Hospitals Campus Northwick Park & St. Mark's Site Level 7W, St Mark's Building
Watford Road Harrow HA1 3UJ
Email;
Tel. +44 (0)20 8869 3494
Fax. 020 8235 4001
Abstract:Dendritic cells (DCs) either boost the immune system(enhancing immunity) or dampen it (leading to tolerance).This dual effect explains their vital role in cancer development and progression. DCs have been tested as a predictor of outcomes for cancer progression. Eight studies evaluated TIDCs as a predictor for colorectal cancer (CRC) outcomes. The detection of tumour infiltrating DCs (TIDCs) has not kept pace with the increased knowledge about the identification of DC subsets and their maturation status. For that reason, it is difficult to draw a conclusion about the performance of DCs as a predictor of outcome for CRC.In this review, we examinecomprehensivelythe evidence for the in situ immune response due to DC infiltration, in predicting outcome in primary CRC and how such information may be incorporated into routine clinical assessment.
Keywords; tumour infiltrating dendritic cells, outcomes, colorectal cancer
Introduction
Colorectal cancer (CRC) is one of the major public health problems worldwide(Siegel, Desantis, & Jemal, 2014). Described as a multi-step disease process, CRC develops slowly over several years and progresses through cytologically distinct benign and malignant states, from single crypt lesions through adenoma, to malignant carcinoma with the potential for invasion and metastasis.Rudolf Virchowfirst described the link between cancer and inflammation, suggesting that the “lymphoreticular infiltrate” at sites of chronic inflammation reflected the origin of cancer. It was not until the last decade that inflammation was accepted as a major factor in the pathogenesis of cancer and some of the underlying mechanisms have been described (Karin, 2006),(Hanahan & Weinberg, 2011). Since these observations, the link between cancer progression and the host’s systemic and localinflammatory response has been further investigated. The inflammatory response is associated with changes in the type, density, and location of immune cells in cancer tumours and has also been linked with weight and lean muscle mass loss (Roxburgh, Salmond, Horgan, Oien, & McMillan, 2009),(McMillan, 2008) (Malietzis et al., 2015). Alsodifferent biochemical or haematological markers have been used in cancer patients to try and quantify the impact of inflammatory response on outcomes (Malietzis et al., 2014).
The use of immunohistochemical (IHC) techniques and animal models has improvedour understanding of both thesystemic and mucosal immune response in CRC. Immune cells possessvariety offunction controlled by complex interactions with tumor-derived factors in the microenvironment(Mantovani, Allavena, Sica, & Balkwill, 2008). Several lines of evidence support the concept that tumour stromal cells are not merely a scaffold, but that they influence growth, survival, and invasiveness of cancer cells, dynamically contributing to the tumor microenvironment, together with immune cells(Grivennikov, Greten, & Karin, 2010). The anomalous phenotype of the tumour can encourage an inflow of inflammatory lymphocytes into tissues around the tumour. This ability of the immune system to function as a prime defence against cancer, to respond, recognise and eradicate any emerging or established tumour areas, acts as an indication of the aggressiveness of the tumour and also as a predictor for disease outcomes (Klintrup et al., 2005),(Väyrynen et al., 2013). Different types of immune cells infiltrate the tumour microenvironment, comprising cells of both the innate and adaptive immune system.
Most tumor cells express antigens that can mediate host CD8+T cells reactions. Cancers that are detected clinically must have evaded antitumor immune responses to grow progressively(Gajewski, Schreiber, & Fu, 2013).Dendritic cells (DCs) are potent antigen presenting cells, capable of sampling antigens and initiating cytotoxic T-lymphocyte response against cancer cells (Banchereau et al., 2000).Colorectal tumor antigens induce DC recruitment, maturation, and cytokine release to generate an effective Th1-type immune response (Cui et al., 2007). However, DCs can also induce tolerance to tumour antigens by promoting anergic T cells(Dudek, Martin, Garg, & Agostinis, 2013). Anergic T cells are functionally inactivated and unable to initiate a productive response even when antigen is encountered in the presence of full co-stimulation(Macián, Im, García-Cózar, & Rao, 2004).Despite their crucial role in generating an immune response, DCs are a heterogeneous and rare type of immune cell. DC subtypes depend on their localization and differ in phenotype and function (Ueno et al., 2010).Their diverse phenotypes allow the various DC subsets to specifically respond to the danger signals they encounter in their microenvironment. Hence, it is not only the presence of a DC but the DC’s explicit subset type and maturation status that will predict the nature of the immune response (Karthaus, Torensma, & Tel, 2012).
Studies mainly using IHC have focused on DCs because of their crucial function in responding to tumour antigens and the presence of tumour-infiltrating DC (TIDCs) correlates to the clinical prognosis for a variety of solid tumours including CRC. But reports studying the role of DC density in patient survival have found contradicting results. We carried out a systematic search of the literature to investigate the hypothesis that TIDCs (identified using IHC techniques) have an impact upon clinical and oncological outcomes in CRC patients.
Methods
Search strategy
This study was performed in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses(PRISMA) recommendations for best practice in reporting systematic reviews (Moher, Liberati, Tetzlaff, & Altman, 2009). The electronic databases Medline, EMBASE and the Cochrane Central Register of Controlled Trials were interrogated with the following search terms: (“Dendritic Cell” OR “DC” OR “DCs”) AND (“colorectal cancer” OR “colon cancer” OR “rectal cancer”). Limits were set from 1st January 1947 to 1st July 2014. Duplicates of citations were removed. A hand-search of reference lists was also performed for additional studies. Two reviewers (GM, GHL) independently assessed all identified abstracts and titles of studies meeting the predetermined selection criteria to confirm eligibility. Papers were retrieved in full if insufficient data was available to make a determination. Disagreements were resolved by third independent review of disputed papers (HO).
Selection criteria
All studies evaluating TIDCs identified through IHC techniques, as a predictor for CRC outcomes or a study with a subgroup analysis that studied TIDCS were included. Conference abstracts were only included if authors provided additional data. Editorials were excluded. Studies were excluded if they did not include patients diagnosed with CRC or other than the TIDCS were studied or no links between TIDCs and CRC outcomes were examined and also if publications were not in English language.
Quality Assessment
The quality of the included studies was assessed according to the Scottish Intercollegiate Guidelines Network (SIGN)(Harbour & Miller, 2001).
Data collection and analysis
The following data were extracted from each study: type of study, patient numbers, stage of the disease, treatment offered, immunohistochemistry details, short and long term survival data [recurrence/ disease-free survival (DFS) and overall survival (OS) and oncological outcomes. The outcome of interest included TIDCs and their effect on postoperative morbidity, recurrence, mortality and additional treatment outcomes.
Results
Search outcomes
Our literature search identified 12 studies matching our selection criteria. Full-text review of these resulted in four exclusions that included three review papersand one study investigating the DCs at the invasive margin of CRC but not associating with outcomes. Eight studies were judged to be eligible in this review, all of which were cohort studies (evidence grade C based on the SIGN grading system)(Ambe, Mori, Enjoji, & Cells, 1989),(Schwaab, Weiss, Schned, & Barth, 2001),(Gulubova et al., 2012),(Nagorsen et al., 2007),(Sandel, Dadabayev, Menon, Morreau, & Melief, 2005),(Nakayama et al., 2003),(Dadabayev et al., 2004),(Liska et al., 2012). A total of 651 specimens, all diagnosed with CRC at all stages, were included. All studies used paraffin embedded stored samples, except one that used AMEX preserved tissue. Different markers were used in order to identify the DC and their various phenotypic subtypes.
Methods used to identify the DCs
The density, type, and activation state of TIDCs have been determined with a variety of different molecules, but S-100 and CD1a were the most widely used markers. Ambeet al.was one of the first studies to describe the presence of S-100 (+) DC s in the gastrointestinal tract (Ambe et al., 1989) . DCs were identified as the S-100+ cells with a dendritic irregular cytoplasm and ovoid nuclei. The same approach was used by Nakayama et al and Liska et al (Nakayama et al., n.d.),(Liska et al., 2012). Two other studies recognised DCs by their distribution pattern and morphology(Sandel et al., 2005),(Dadabayev et al., 2004). DCs were identified as the HLA-DR (+)/ CD83 (+) positive cells by Schwaabet al(Schwaab et al., n.d.). Cells stained with S-100, HLA-DR, CD1a or CD83 were considered to be DCs by Gulubovaet al(Gulubova et al., 2012). Finally Nagorsenet al. classified DCs as S100+/CD163+ double stained cells (Nagorsen et al., 2007). Table 1 shows the demographics and DC identification methods employed.
DC Populations studied
Few studies haveaimed to specifically detect the infiltration of tumours by the distinct subsets. Two studies have endeavoured to assess the maturation status of TIDCs by staining for CD83, a DC-specific maturation marker expressed by all mature subsets (Ambe et al., 1989),(Gulubova et al., 2012),.Two studies have also used molecules such as DC-LAMP and DC-SIGN as maturation markers (Nagorsen et al., 2007),(Sandel et al., 2005).DC-SIGN is expressed by immature DCs, whereas DC-LAMP is present on mature DCs. Also Schwaabet al. focused not only on the presence of mature TIDCs but also on their activation status staining for CD40 surface marker (Schwaab et al., 2001).
DC Location identification
Six of the selected studies identified DCs in the tumour margin area (Ambe et al., 1989),(Schwaab et al., 2001),(Gulubova et al., 2012),(Nagorsen et al., 2007),(Sandel et al., 2005),(Nakayama et al., n.d.),(Dadabayev et al., 2004),(Liska et al., 2012).Gulubova et al and Nagorsen et al included tumour stroma and tumour epithelium (Gulubova et al., 2012),(Nagorsen et al., 2007). The latter study also assessed the tumour epithelium for the presence of TIDCs.
Impact of TIDCs on CRC outcomes
The presence of density staining for S-100 (+) cells in the tumour margin area was associated with survival outcomes in four studies with contradictory results (Ambe et al., 1989),(Sandel et al., 2005),(Nakayama et al., 2003) (Liska et al., 2012). In the study from Ambe et al, DCs were identified using anti-S100 antibody and the patients with high DC count had a significantly improved 5-year OS compared to those with low DC count (70% Vs 33%; P<0.001). The S-100 (+) cell count was significantly lower in CRC patients who had recurrence (P=0.003) or poor survival (P=0.001) from the Nakayama et al study. S-100 (+) cells in the tumour margin were not associated with poorer survival in the study by Sandelet al. (Sandel et al., 2005). Positivity for S-100 (+) cells had no significant influence on OS or DFS in the study of Liska et al. (Liska et al., 2012). Two studies reported that the increased presence of these S-100 (+) cells in the tumour stroma had a positive effect on the OS in the patients studied. The 5-year OS survival for the group with the high count was 65% compared to 35% for the low count (P=0.02) in the Gulobova et al study whereas for the Nagorsen et al study was 60% Vs 40% (P=0.04) respectively (Gulubova et al., 2012)(Nagorsen et al., 2007).
Three studies investigated the impact of the maturation status of the TIDCs on CRC outcomes with conflicting results. Increased CD208 (+) cells in the tumour margin had a negative impact on OS in the study by Sandelet al (5y OS 60% Vs 25%; P=0.004); whereas high CD83 (+) (5-year OS 60% Vs 40%; P=0.04) and CD83 (+)/CD40 (+)(RR 0.24; 95%CI 0.03-1.6) cells from Gulubovaet al. and Schwaabet al, respectively, had a beneficial effect on the OS. (Sandel et al., 2005),(Gulubova et al., 2012),(Schwaab et al., 2001). Table 2 summarizes the different subsets of DCs studied, the location of assessment and the impact on CRC outcomes.
Discussion
Our literature review shows that an increased infiltration of activated and mature DC in colorectal cancersmay be associatedwith improved OS and DFS, implicating TIDC’s essential role in localdisease control.Patients with increased TIDCs, identified as S-100 (+), in the tumour margin and the tumour stroma reported to have a 5-year OS of up to 70% compared to patients with decreased TIDCs, in whom the 5-year OS varied between 35-40% in studies identified. High numbers of mature TIDCs was another factor suggested to be associated with better prognosis in two of the studies with 5-year OS up to 60% compared to 40 % in patients with low numbers. In addition, an increased infiltration of DCs in colorectal tumour tissue corresponded to increased recruitment of CD45 (+) lymphocytes including CD4 and CD8 T cells (Dadabayev et al., 2004). This finding provides further evidence that for a successful anti-tumour immune response to occur, TIDC recruitment is essential as it corresponds to an increased infiltration of cytotoxic T-lymphocytes.
Controversy, however, was noted in the method of identifying TIDCs from IHC specimens, and in the correlations between the presence of TIDCs and clinical prognosis. A common limitation described in selected studies was the identification method for DCs, thus the cell morphology and distribution pattern was the method of preference in the majority of the studies. Thus far, the classical markers, namely, CD1a, S-100, CD83, and DC-SIGN, used for TIDC identification do not reveal unambiguous correlations with cancer progression. The reason behind the above observation can be due to the fact that the there is no specific marker expressed exclusively on these cells. For example, S100 proteins are also expressed on a variety of cells of myeloid origin including granulocytes, macrophages and epithelial cells during inflammation(Foell, Wittkowski, Vogl, & Roth, 2007); CD1a is also expressed on macrophages.(Valladeau & Saeland, 2005) These molecules (CD1a, S-100) can be used in conjunction to detect tissue resident interstitial DCs. Immunohistochemical analysis of CRC samples by Sandel et al showed a negative correlation between the number of mature TIDCs and a favourable clinical outcome. This observation was in contrast to other findings described from other studies. A possible explanation of this discrepancy could be due to the decreased immunogenicity of the included CRC tumours in the above study. Microsatellite-instable tumours are characterized by the presence of increased infiltration of immune cells and are associated with better overall prognosis (Greenson et al., 2009) . The differences in quantity and type of TIDCs can be a reflection of this phenomenon.
However, it is important not just to associate outcome with the presence or absence of TIDC, but also as some studies demonstrated that the outcome of CRC further correlated with activation/maturation status. Immature DC are highly potent at acquiring antigen, but are less effective in presenting antigen to naïve T cells in corresponding lymph nodes and may induce tolerance (Al-Hassi et al., 2014) . Therefore, for a successful anti-tumour immune response to occur, TIDC must be activated and should have undergone maturation, migrated to the lymph nodes and stimulated tumour-specific lymphocyte recruitment and survival (Iwamoto et al., 2003). There was better overall survival in patients with TIDCs showing high HLA-DR expression and CD83, emphasising the importance of determining the phenotype of TIDC(Gulubova et al., 2012).
It was also suggested that TIDCs can induce immune tolerance that can lead to further progression of CRC despite intact immune response, the so-called “immune escape”. Nagorsen et al extensively characterised TIDC and other immune cells in close interaction with DC. This study further showed that there was significantly increased infiltration of DC in tumour without systemic T-cell response and increased infiltration of regulatory T cells. It is interesting to note that despite TIDCs inducing mostly tolerogenic immune response, there was still overall improved outcome in patients with increased TIDC.
Mechanism by which TIDC may influence DFS and OS is complex, but it can be attributed to the host’s immune response to tumour. In the studies identified in this review, the host’s response can be associated with either the local microenvironment or systemic effect of tumour. As tumour progresses, it alters the cellular components of various immune cells (T-cells, NK cells) and dysregulates cytokine levels influencing the density, activation and maturation status and migration ability of DCs (Naito et al., 1998)(Conti & Thomas, 2011). Consequently, this has a systemic effect on the characteristics of circulating DCs leading to immune dysfunction, which will correlate with DFS and OS of patients with CRC (Lee et al., 2014).
There are several limitations to our study. The number of studies is small and with limited patient numbers. Studies have varying designs and quality; hence evaluation of aggregated data was unsuitable. All were retrospective cohort studies from single institutions. Hence no causal relationship between TIDCs and CRC outcomes can be truly ascertained. Study heterogeneity made explanation of the data difficult. What is clear is that further and more rigorous studies are required to validate a widely accepted protocol for assessing TIDCs from CRC pathological specimens.
Conclusion
In summary, TIDCs in colorectal tumours showed a distinct infiltration pattern based on their phenotypic status. The presence of TIDCs may be associated with better long-term outcomes for patients with CRC. TIDCs identified through IHC techniques in patients undergoing treatment for CRC may play a role in identifying patients with potentially poorer prognosis and may facilitate clinical decision-making. Evidence is evolving for TIDCs prognostic utility but to date no specific therapies or interventions to modify pre- and postoperative inflammatory responses exist. Hence the role of DCs in the local immunological response of the host in controlling the growth and progression of CRC requires greater clarification and may also be a potential therapeutic target.
Acknowledgments: None
Declaration of interest: None
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