Introduction

Over the past 10 years, accumulating evidence has indicated that a pronounced intratumour inflammatory reaction plays a crucial role in host response to cancer. Various inflammatory elements (dendritic cells, macrophages, granulocytes and lymphocytes) have been found in the tumour micro-environment, both in supporting stroma and among epithelial cells in tumour nests.

The presence of tumour-infiltrating lymphocytes (TILs) has been shown to correlate with a favourable prognosis in several human malignancies such as melanoma and ovarian, prostatic, renal-cell, breast and colorectal carcinomas [12, 25, 27, 38, 42]. The presence of abundant lymphocytic tumour infiltration is a peculiar histological feature common to gastric carcinomas (GCs), both with Epstein–Barr virus (EBV) infection [4, 28, 37] and with a high level of microsatellite instability (MSI) [4, 34].

The immune response to cancer cells may be mediated by non-specific natural killer (NK) cells [30] or by cancer-specific lymphocytes, which are usually CD8-positive [2, 31].

In GCs, a high level of NK infiltration was significantly associated with a better prognosis of disease [14].

In MSI colorectal carcinomas, the intratumour lymphocytes have been demonstrated to be prevalently cytotoxic-activated T cells [7]. Their presence could be responsible for the increased apoptosis of tumour cells observed in these tumours, although Michael-Robinson et al. [23] demonstrated that TILs and apoptosis are independent characteristics of MSI tumours.

In the present investigation, we studied a series of GCs characterised for MSI status and EBV infection with the aims of clarifying whether TILs are associated with a better clinical outcome in GC patients and of defining the nature and the activation status of TILs in the different subtypes of GCs.

Materials and methods

Case selection

The study included 96 cases of sporadic advanced GCs (Table 1) operated on at the University Hospital of Varese between January 1980 and June 1999. The majority of the tumours were from a series of 185 cases which had previously been examined for the presence of high MSI, expression of hMSH2 and hMLH1 proteins and EBV infection with in situ hybridisation for EBER-1 [4]. In particular, 35 MSI and 31 microsatellite stable (MSS) GCs were selected for the presence of abundant lymphoid infiltration, while 20 cases, showing a less abundant lymphoid infiltrate, were selected from the remaining consecutive MSS glandular carcinomas. Ten other cases were added to the series because they were known to be EBV-positive. Altogether, 35 cases were MSI, and 61 were MSS carcinomas, 17 of which were positive for EBV.

Table 1 Clinico-pathological data of 96 gastric carcinomas
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The tumours were classified, according to the criteria outlined by Carneiro et al. [3], as glandular, solid or mixed types and according to the Lauren criteria [19]. The tumour stage was assessed using the tumour node metastases (TNM) system defined by the International Union Against Cancer [35]. The clinico-pathological data of the GCs, characterised for MSI status and EBV infection, are summarised in Table 1.

All patients were followed-up either until death or for a median period of 150 months (range 42–238 months); information was obtained from the Lombardy Tumour Register and from the record offices of local authorities. The mean overall follow-up was 64.3 months (range 0–238).

Immunohistochemical study

The characterisation of TILs was performed on formalin-fixed, paraffin-embedded materials using the avidin–biotin–peroxidase complex (ABC) methods. Briefly, sections were deparaffinised, rehydrated and, for selected antigens, pre-treated with different antigen-retrieval solutions in a domestic 750-kW microwave oven (see Table 2). Endogenous peroxidase activity was quenched in 3% H2O2 in water for 10 min. Primary antibodies (listed in Table 2) were applied overnight at 4°C. Sections were then incubated with biotinylated anti-mouse immunoglobulins and with ABC peroxidase complex, each for 1 h at room temperature.

Table 2 List of primary antibodies used
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Immunoreactive intraepithelial lymphocytes were counted at 400× (Leitz, Laborlux K; field area 0.173 mm2) in ten consecutive fields, selecting areas containing the maximal number of neoplastic cells with minimal reactive stroma and necrosis. The mean value of immunoreactive TILs per high-power field was reported. Only the immunoreactive lymphocytes in direct contact with tumour cells were included in the count.

The apoptotic index was expressed as the percentage of M30 immunoreactive cells per 2,000 tumour cells counted in the ten most positive fields at 200×. Areas of intraglandular or superficial necrosis were not considered for the evaluation of the apoptotic index.

Statistical analysis

The statistical significance of the results was evaluated using the Wilcoxon rank-sum test for unpaired data, chi-square test and Fisher's Exact Test. The correlation of patient survival with the immunohistochemical and clinico-pathological data was estimated using the Kaplan–Meier product limit method, and statistical differences were tested using the log-rank test. A multivariate analysis was performed with the Cox proportional-hazard regression model.

Results

Clinical and pathological features

The clinico-pathological data of the GCs are summarised in Table 1. There were slightly more males (male to female ratio 2.4:1) with MSS/EBV+ cancers compared to MSI and MSS/EBV− cancers (male to female ratios 1.5:1 and 1.2:1, respectively).

Both MSI and MSS/EBV− carcinomas were more frequent in the antrum (77 and 68%, respectively), whereas MSS/EBV+ carcinomas were prevalently located in the gastric fundus (47%) and in the gastric stump (29%) (p<0.001).

Tumour size varied in diameter from 20 to 100 mm. The mean diameters were 58.6, 60.3 and 39.7 mm for MSI, MSS/EBV+ and MSS/EBV− carcinomas, respectively.

Histologically, 64% of all tumours were glandular carcinomas. In particular, 74% of MSI and all the MSS/EBV+ cases were poorly differentiated, whereas 61% of MSS/EBV− were moderately differentiated carcinomas (Fig. 1). According to Lauren's criteria [19], 80% of tumours were intestinal, and only 4% were diffuse GCs; 16% of cases were unclassified.

Fig. 1
figure 1

Gastric carcinoma with microsatellite instability (a), EBV infection (b) and without microsatellite instability and EBV infection (c) (haematoxylin and eosin stain, original magnification ×200)

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At diagnosis, MSS/EBV− and MSS/EBV+ carcinomas showed lymph node metastases in 31 of 43 (72%) and 14 of 17 (82%) cases, whereas they were present in only 18 of 35 (51%) MSI carcinomas (p<0.05).

Fifty (52%) patients died of disease after an average time of 32.5 months (range 2–181), including 14 (40%) patients with MSI, 27 (62%) with MSS/EBV− and 9 (53%) with MSS/EBV+ carcinomas.

Thirty (31%) patients died of other causes (mean follow up 46.5 months, range 0–212), and only 16 patients (17%) were still alive at the last follow-up after a mean time of 150 months (range 42–238).

The survival curves (Fig. 2) showed a significantly different prognosis in the three groups (p<0.05). In particular, the overall survival was significantly higher for patients with MSI than with MSS/EBV− GCs (p=0.01). The mean 5-year survival was 59, 46 and 32% for MSI, MSS/EBV+ and MSS EBV− GCs, respectively.

Fig. 2
figure 2

Failure time according to MSI and EBV status [log-rank chi-square 6.35 (p=0.0417)]

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Characterisation of lymphoid infiltration

Lymphocytes were present both within tumour-cell nests and in peritumoural stroma. CD3+ intraepithelial TILs varied in number from 0 to 254 in a high-power microscopic field (mean value 31.6, median value 14.9).

The mean number of CD3+ TILs was 30.7 (range 1.6–77), 6.6 (range 0–30.4) and 100 (range 43.3–254) in MSI, MSS/EBV− and MSS/EBV+, respectively, and the differences were statistically significant (p<0.001; Fig. 3).

Fig. 3
figure 3

CD3 immunoreactive tumour-infiltrating lymphocytes in MSI (a) and MSS/EBV− (b) gastric carcinomas (immunoperoxidase; original magnification ×200)

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The nature and the activation status of TILs were analysed in a subset of GCs, selected because sufficient material was available for study, and the immunohistochemical results are summarised in Table 3. The majority of TILs showed an intense CD8 immunoreactivity. CD8+ TILs varied from 0.3 to 118.7 cells per field, the mean value being 25.5 and the median value being 14.6. The mean numbers of CD8+ TILs in MSI (21.7; range 2.1–56) and in MSS/EBV+ (69.6; range 26–118.7) GCs were significantly higher (p<0.001) than those found in MSS/EBV− cases (6.4; range 0.3–23.7).

Table 3 Immunophenotype of TILs and apoptosis of tumour cells in 72 gastric carcinomas
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A minimal number of TILs was composed of NK cells. The number of CD57+ TILs was significantly higher (p<0.01) in MSS/EBV+ (mean value 4.2) than in MSS/EBV− (mean value 0.8) or in MSI (mean value 3.1) GCs.

The presence of cytotoxic cells was confirmed by the expression of T-cell intracellular antigen (TIA-1) in the cytoplasmic granules in a considerable proportion of TILs. The mean number of TIA-1+ TILs was significantly higher in MSS/EBV+ (32.05; range 3–84.6) than that observed in MSS/EBV− (5.2; range 0–29.1; p<0.001) and MSI (16.7; range 0.4–59.5; p<0.05) GCs.

The activation status of TILs was assessed on the basis of immunoreactivity for granzyme B and perforin. The number of granzyme B+ TILs (Fig. 4) was significantly higher (p<0.001) in MSI and in MSS/EBV+ than in MSS/EBV− GCs (mean values 7.5 and 8.6, respectively, vs 0.8). Perforin+ TILs were also significantly more numerous in MSI GCs and in MSS/EBV+ than in MSS/EBV− tumours (mean values 5.9 and 9.2, respectively, vs 0.9; p<0.001 and p<0.05).

Fig. 4
figure 4

Granzyme B immunoreactive tumour-infiltrating lymphocyte in EBV+ gastric carcinoma (immunoperoxidase, original magnification ×400)

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Apoptosis of tumour cells

To assess the apoptotic index of tumour cells, GCs were immunostained with the M30 CytoDEATH antibody that recognises a fragment of cytokeratin-18 cleaved by a caspase at the beginning of the apoptotic process. Tumour cells showed an intense and variable cytoplasmic M30 immunoreactivity in relation to the apoptotic stage of the cell (Fig. 5). At the beginning of the apoptotic process, in cells which were still histologically normal, the immunoreactivity was distributed throughout the cytoplasm, whereas in damaged cells, the immunoreactivity was localised in granules, which varied in size and number, within the cytoplasm.

Fig. 5
figure 5

Apoptotic cells immunoreactive for M30 CytoDEATH antibody in a microsatellite-instable gastric carcinoma (immunoperoxidase, original magnification ×400)

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The distribution of M30 immunoreactive tumour cells differed among the various tumours; in some cases, M30+ cells were dispersed throughout the tumour, while in other cases, they were localised near necrotic areas or within the glandular lumens. The mean percentage of M30+ cells varied from 0.05 to 29.2% (Table 3). M30+ cells were more frequent in MSI and in MSS/EBV+ than in MSS/EBV− GCs (mean percentages 5.9 and 2.9, respectively, vs 2.3; p<0.01).

Necrotic areas and intraglandular abscesses, frequently observed in MSS/EBV+ GCs, were always M30-negative.

Correlation with survival

A univariate analysis (Table 4) revealed that the low-tumour stage (p<0.0001), the absence of node metastases (pN, p<0.0001), the low depth of tumour invasion (pT, p<0.0003), the intestinal type (p=0.04), the presence of MSI (p=0.02) and high numbers of CD3+ TILs and CD8+ TILs correlated with survival. In particular, carcinomas with CD3+ TILs higher than 14.9 (Fig. 6) and CD8+ TILs higher than 9.5 (Fig. 7) were associated with a significantly improved survival (p=0.01 and p<0.05, respectively).

Table 4 Relationships of age, sex, Carneiro and Lauren classification, tumour site, pT, pN, stage, EBV status, MSI status, diameter, CD3+ TILs and CD8+ TILs with survival
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Fig. 6
figure 6

Failure time according to CD3+ TILs [relative risk 2.03; log-rank chi-square 6.38 (p=0.0115)]

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Fig. 7
figure 7

Failure time according to CD8+ TILs (relative risk 2.09, log-rank chi-square 4.92 (p=0.0265)]

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The presence of intratumoural CD8+-activated lymphocytes (perforin >1.5 and granzyme B >1.75) was also statistically significant (p<0.01).

On the basis of a Cox regression analysis, only a low tumour stage (p<0.00001) and a high number of CD3+ TILs (p=0.02) were identified as independent prognostic factors (Table 5).

Table 5 Multivariate Cox proportional-hazard analysis of overall survival
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Discussion

Abundant TILs seem to be associated with a more favourable prognosis in various malignancies, including melanomas and breast, renal, colorectal and GCs [12, 25, 27, 38, 42]. Tumour-associated lymphocytes show oligoclonal expansion [13], exhibit tumour-specific cytotoxic activity in vitro [32] and recognise tumour-specific antigens [17, 29, 33].

The prognostic significance of lymphocytic infiltration in gastric cancer was first emphasised by MacCarty and Mahle [21]. In 1976 Watanabe and colleagues [39] observed a high survival rate among patients with GCs with lymphoid stroma. With the aim of evaluating the prognostic significance of TILs, we selected, from a series of consecutive GCs, those showing abundant lymphoid infiltration. The MSS/EBV− and MSS/EBV+ groups were enriched in number to be better comparable. Our study, with the limit that our cases were partly selected, seems to indicate that the presence of intratumoural T cells correlates with the clinical outcome of advanced gastric cancer after apparently radical surgery. In a series of 96 patients with advanced GCs, cancer-specific survival of patients appeared to be increased by a factor of approximately 2 if more than 14.9 CD3+ TILs or more than 9.5 CD8+ TILs were present per high-power microscopic field. A univariate analysis showed that, in addition to clinico-pathological parameters including stage, lymph node status, depth of tumour invasion, histological type and MSI status, TIL count was significantly correlated with patient survival. A Cox regression analysis confirmed TILs as an independent prognostic factor together with stage.

Two types of GCs are characterised by a large number of TILs: the MSI- and the EBV-associated GCs [16, 28, 34, 37].

Like patients with sporadic MSI colorectal cancers [8, 11, 40], patients with sporadic MSI GCs apparently have a better survival rate than patients with MSS tumours [34, 41]. EBV-positive GCs have sometimes been associated with a survival advantage [5, 16, 36]. However, in 1994, Nakamura et al. [26] observed that, among GCs with lymphoid stroma, there was no significant difference in the 5-year survival rate between cases with EBV-positive and EBV-negative GCs.

The molecular characteristics of tumours that elicit an enhanced immune response are unclear, but the increased production of abnormal peptides seems to be the more probable hypothesis to explain this phenomenon. In patients with MSI colorectal carcinomas with abundant T-cell infiltration, Ishikawa et al. [15] demonstrated the production of specific antibodies against an abnormal fragment of CDX2 protein found in tumour tissue. The aberrant protein is produced by a frameshift mutation in the microsatellite sequence of the CDX2 coding region. This study seems to support the hypothesis that, in MSI tumours, the alteration of the mismatch repair system is responsible for the production, by tumour cells, of abnormal tumour-specific peptides which recruit lymphocytes in the tumour and induce an immune response.

A similar mechanism can operate in EBV-positive tumours, where the recruitment of TILs could be the consequence of the production by a virus, via tumour cells, of abnormal peptides, although this hypothesis has not been demonstrated yet. As a matter of fact, in their paper, Kuzushima and colleagues [18] observed that CD8-infiltrating lymphocytes did not recognise EBNA−1 and BRAF antigens produced by tumour cells.

The nature and the activation status of TILs in our cases of GCs seem to be similar to that observed by Dolcetti et al. [7] in MSI colorectal carcinomas. In both MSS/EBV+ and MSI GCs, the majority of TILs was represented by cytotoxic CD8+ cells. A minor component of TILs was represented by NK CD57+ cells. Cytotoxic effector CD8+ and NK cells are characterised by the presence of TIA-1 immunoreactive granules in their cytoplasms, independently of their activation status. TIA-1 protein is crucial for DNA fragmentation and the apoptotic process [1]. In our study, the mean value was significantly higher in MSS/EBV+ and MSI than in MSS/EBV− GCs.

The production and secretion of granzyme B and perforin are indispensable for the activation of cytotoxic and NK lymphocytes. In the presence of Ca ++, the monomers of perforin are released by the killer lymphocytes and inserted into the membrane of target cells, where they form membrane pores which promote the entry of cytolytic enzymes, in particular, TIA-1 and granzyme B [20].Granzyme B is a granule-associated serine protease that triggers the apoptotic cascade, probably by activating caspases 10, 3 and 7 [9].

Both perforin+ and granzyme B+ TILs were significantly more numerous in MSS/EBV+ and MSI than in MSS/EBV− GCs, suggesting that the activation of cytotoxic cells is a specific event priming the apoptotic process in MSS/EBV+ and MSI GCs.

The M30 monoclonal antibody identifies epithelial apoptotic cells and has been previously used by Michael-Robinson et al. [24] to evaluate the apoptotic index in MSI colorectal carcinomas. This antibody binds to the caspase-cleaved fragment of cytokeratin-18 during the early steps of epithelial cell apoptosis. The demonstration that apoptosis of neoplastic cells was significantly more frequent in MSI (5.87%) and in MSS/EBV+ (2.95%) than in MSS/EBV− (2.26%) GCs supports the hypothesis that, in these tumours, TILs play a crucial role in the increased apoptotic cell death of neoplastic cells.

As for the difference between MSI and MSS/EBV+ GCs, it is worth noting that, although CD3+, CD8+ and TIA-1+ TILs were significantly higher in MSS/EBV+ than in MSI GCs, the percentage of activated TILs, as well as the apoptotic index of tumour cells, were not significantly different between the two subsets of GCs. These data might explain the absence of a significant difference in prognosis between patients with MSS/EBV+ and MSI GCs.

In our study, the prognosis of MSS/EBV+ cases appears to be better than that of MSS/EBV− cases, although the difference is not statistically significant. In addition, the prognosis of patients with MSI+ GCs appears to be better than that of patients with MSS/EBV+ GCs. Previous studies examining the correlation between survival and EBV infection in GCs did not consider the MSI status [5, 16, 22] with the exception of that of Grogg et al. [10]. In this context, it is important to underline that in our study, all the MSI GCs are EBV-negative, in agreement with the findings of previous investigations [6, 10]. In addition, our results confirm that MSI GCs are associated with a significantly better prognosis than that of the MSS GCs, and particularly, of MSS/EBV− GCs.

In conclusion, our study demonstrates that the presence of a high number of CD3+ TILs is a favourable prognostic factor, independently of the pathogenesis of GCs. EBV-positive and MSI GCs are two subsets of tumours characterised by a similar intratumour lymphocytic infiltration prevalently composed of activated cytotoxic CD8+ cells. The relatively better prognosis of these subsets of GCs seems to be related to the increased host immune response against abnormal peptides, probably produced by the virus infection or by the presence of an altered mismatch repair system.