Stromal regulatory T-cells are associated with a favourable prognosis in gastric cancer of the cardia

Gastric carcinoma is the third most common gastrointestinal tumour type in developed countries [1]. Similarities in risk-factors for adenocarcinoma of the gastric cardia and the oesophagus [2] together with the stable incidence of cardiac gastric cancer despite an overall decline of gastric malignancies [3] suggest that these tumours should be separated from distally located gastric adenocarcinomas [4].

Escape from immunosurveillance is a fundamental step in cancer development. There is accumulating evidence indicating that CD4⁺CD25⁺FoxP3⁺ regulatory T-cells (Treg) are able to induce tolerance to self-antigens and may also inhibit anti-tumour immune response. Tregs can inhibit effector T-cells, suppress the antigen presenting function and promote the immunosuppressive function of dendritic cells, monocytes and macrophages [5, 6].

The prognostic significance of tumour infiltrating lymphocytes (TIL) in cancer therapy is not fully understood. A beneficial prognostic effect of increased lymphocyte infiltration has been shown for various tumour types. In ovarian carcinoma both intratumoural CD3⁺ and CD8⁺ TIL were associated with improved survival [7, 8] while Treg were identified as a significant negative prognostic factor [9]. An inverse effect was observed in anal cancer where low amounts of CD3⁺ and cytotoxic TIL were prognostically favourable, while Treg presence had no influence on prognosis [10]. A recent investigation of gastric malignancies found no correlation between the number of Treg and patient survival, but emphasized the significance of their distribution pattern [11].

We investigated the prognostic value of infiltration by various TIL-subgroups and macrophages including Treg in adenocarcinoma of the cardia in both primary tumour and draining lymph nodes.

Tregs inhibit the function of effector cells [5, 6]. Consequently, an inhibitory effect of Treg on anti-tumour immunity has been predicted. In agreement with this hypothesis, large numbers of tumour-infiltrating Treg were shown to be associated with poor outcome in ovarian carcinomas [9]. However, studies of other tumours have produced conflicting results. Thus, no prognostic effect of Treg was observed in a study of anal carcinomas [10]. In certain lymphomas, tumour-infiltrating Treg were associated with favourable prognosis [14]. A recent study of FoxP3⁺ Treg in colorectal cancer also showed high Treg numbers to be associated with improved survival [15].

Studies on gastric carcinomas have also yielded conflicting results. Kono et al. found a higher percentage of Treg among PBMCs in patients with advanced gastric carcinoma. Higher numbers of Treg in PBMCs had an adverse effect on outcome, and numbers of Treg declined to normal values following curative resection [16]. Mizukami et al. found no effect of Treg counts on survival but showed that a diffuse dissemination of Treg was associated with poor survival while accumulation of Treg in the peritumoural region correlated with a more favourable outcome. But the former distribution pattern was associated with diffuse gastric cancers while the latter was predominantly found in intestinal type carcinomas [11]. Kawaida et al. found that tumour draining lymph nodes showed an increased prevalence of CD4⁺CD25⁺ TIL as compared to control mesenteric nodes. However, no specific correlation between survival and intensity of Treg infiltration could be demonstrated [17]. These previous studies have analyzed TIL numbers in different compartments ranging from primary tumour to PBMC, thus making comparison of results difficult.

For these reasons, we have decided to focus on a morphologically homogeneous group of intestinal-type adenocarcinomas located at the gastric cardia. We have assessed the numbers of tumour-infiltrating immune cells in different tumour-related compartments in relation to outcome parameters. We show firstly that the presence of various TIL subsets including Treg, cytotoxic T-cells as well as tumour-infiltrating macrophages within the epithelial compartment had no significant influence on survival rates. This is in contrast to our previous analysis of anal carcinomas [10]. This may be attributable to the differing growth patterns of the two tumour entities with anal carcinomas growing in a more solid fashion with less stromal support as compared to gastric adenocarcinoma. We identified only two parameters which were of prognostic significance. High numbers of Treg in the tumour stroma at the primary site were associated with improved prognosis while this was not the case for other tumour-related compartments. Furthermore, high numbers of macrophages in combination with low numbers of Treg in the tumour stroma indicated poor outcome. Moreover, we show that the number of Tregs as a proportion of total T-cells is higher in both in tumour stroma and in affected lymph nodes as compared to uninvolved lymph nodes. This enrichment of Tregs suggests that these cell are actively recruited to the tumour site and points to a role for Tregs in inhibiting local immune reactions by direct cell-to-cell contact [18].

On the face of it, these findings are unexpected and contrast previous studies of ovarian carcinoma [7‐9, 19]. Nevertheless, our findings parallel recent studies of Treg in colorectal cancer [15]. It is important to keep in mind that Treg exert their function via a direct membrane contact mechanism [18]. This might explain the observation that a larger proportion of T cells are regulatory T cells both in the tumor stroma and the affected lymph nodes as compared to the control lymph nodes.

Chronic inflammation, either related to H. pylori infection or to autoimmune gastritis, is considered the major risk factor for the development of gastric cancer of intestinal type. This is probably also true for gastric adenocarcinoma located at the cardia [20]. Development of chronic H. pylori gastritis and progression to gastric atrophy and preneoplastic lesions has been shown to be associated with the development of a Th1-predominant local immune reaction in mouse experiments [21]. Thus, the presence of large numbers of local Treg may well help to dampen these local inflammatory responses. This notion is also supported by the observation that gastric Treg can suppress H. pylori-induced T cell proliferation and IFN-gamma production [22]. It is speculated that Treg may inhibit local effector T-cells contributing to gastric carcinogenesis. A possible mechanism may be that the effect of Treg is mediated via a down-regulation of harmful proinflammatory cytokines known to promote malignancy [23‐25]. The notion that Treg may exert an inhibitory effect on gastric cancer development is also in keeping with our observation that Treg are particularly prevalent in earlier stages of gastric carcinogenesis since any effect of these cells is likely to be greatest at this time point. Nonetheless, further functional studies will be necessary to elucidate the exact role of Treg in gastric cancer. Moreover, there is the emerging concept that not all FoxP3 positive cells might be natural Tregs with stable FoxP3 expression. Natural Treg cells might be detected by analyzing the Treg-cell specific demethylated region (TSDR) in tissue specimen [26].

We also find that a higher number of Granzyme B positive T cells in uninvolved lymph nodes is associated with a better outcome. Granzyme B is a key cytolytic mediator secreted by activated cytotoxic T cells. A large systemic number of activated cytotoxic T cells as represented by large numbers in uninvolved lymph nodes may be an indicator of an efficient systemic host anti-tumor immunity [27].

Moreover, we show that large numbers of tumour-associated macrophages in conjunction with small numbers of Treg are associated with poor prognosis. This is in agreement with a previous study [28]. It is now well accepted that tumour-associated macrophages promote tumour development for example through stimulating angiogenesis or modulating the plasticity of extracellular matrix [29]. Moreover, it has been shown recently that activated macrophages may activate Wnt signalling in gastric cancer epithelial cells via TNF-alpha thus contributing to tumour development [30].

Our results suggest that inflammatory processes within the tumour stroma of intestinal-type adenocarcinoma of the gastric cardia may affect outcome in two ways. Tumour-infiltrating macrophages are likely to promote carcinogenesis as has been reported previously for various tumour models. By contrast, Treg may down-modulate local inflammatory processes which promote carcinogenesis. Thus, inhibition of Treg may not be a feasible treatment option in gastric adenocarcinoma.