Introduction
Lynch syndrome (LS), or hereditary nonpolyposis colorectal cancer (HNPCC), is characterised by an autosomal dominant inheritance of early-onset colorectal cancer and an increased risk of other cancers, in particular cancer of the endometrium [
1]. LS is caused by germline mutations in a mismatch repair gene, mostly hMLH1, hMSH2 and hMSH6 [
1]. These genes encode proteins involved in DNA repair. Failure of the DNA mismatch repair machinery to repair errors occurring during DNA replication leads to increased length variation of simple, repetitive sequences distributed throughout the genome, so-called microsatellite instability (MSI). An international set of markers is developed to test for MSI. Tumours are scored MSI-high if at least 30% of the markers show instability, MSI-low if less than 30% show instability, or MS-stable (MSS) if none of the markers shows instability [
2]. The MSI-H phenotype is found in up to 92% of colorectal cancers associated with LS and in 10–15% of sporadic colorectal cancers [
1].
Dysfunction of one of the mismatch repair genes results in a rapid accumulation of genetic alterations in susceptible genes, including genes involved in growth suppression, apoptosis and signal transduction [
1]. These alterations contribute to an accelerated adenoma–carcinoma sequence in LS compared to sporadic cancer. This is illustrated by the relative frequent occurrence of interval cancers between regular surveillance colonoscopies [
3,
4]. In addition, LS adenomas frequently harbour a villous component and high-grade dysplasia, both markers of increased cancer risk [
5,
6]. Among the genes that are targeted in the setting of MSI are
BAX and
caspase 5, suggesting that disturbances in apoptosis regulation may be important as a mechanism behind the accelerated colorectal carcinogenesis in LS [
7,
8].
Apoptosis can be either initiated by the cell itself or by certain external stimuli. These external stimuli may induce apoptosis by targeting one of two pathways [
7]. The ‘extrinsic’ pathway is initiated by triggering cell death receptors on the cell surface, leading to activation of the intracellular apoptotic machinery. The ‘intrinsic’ pathway of apoptosis is initiated via the mitochondria by cellular stress, such as chemotherapeutic drugs and radiation. Fas is a protein of the tumour necrosis factor (TNF) receptor superfamily, known to induce apoptosis through the extrinsic pathway in sensitive cells by binding to its natural ligand, Fas Ligand (FasL). In the normal human colon, all epithelial cells express Fas, but not FasL [
9]. In contrast, many colorectal carcinomas and some adenomas show FasL expression [
10‐
13]. It has been hypothesized that FasL expression in tumour cells might enable these cells to evade immune destruction by inducing apoptosis in tumour-infiltrating lymphocytes (TILs), a concept which has become known as the Fas counter-attack [
14]. In a previous study, FasL expression was reported to be higher in MSI-H colorectal cancers compared to MSI-L and MSS cancers [
15]. To date, FasL expression has not been examined in colorectal tumours from LS patients. We hypothesized that a diminished Fas counterattack between LS and sporadic colorectal tumours play a role as a mechanism to explain the difference in carcinogenesis. Therefore, FasL expression, tumour cell apoptosis and number of TILs in colorectal neoplasms from LS patients in comparison with sporadic cases were studied.
Discussion
In this study, we sought to determine whether the Fas counterattack plays a role in the accelerated colorectal carcinogenesis in LS patients compared to sporadic disease. We found no support for such a mechanism, as FasL expression was similar between LS and sporadic tumours. In addition, there were no correlations between the percentages of FasL positivity and the number of TILs. A novel finding from our study was that LS-associated adenomas, in addition to LS-associated carcinomas, are also characterized by higher numbers of TILs compared to sporadic cases.
Colorectal carcinogenesis in LS is believed to be accelerated when compared with sporadic cases. This is illustrated by the relative frequent occurrence of interval cancers between regular surveillance colonoscopies. In addition, LS-associated adenomas frequently harbour villous components and high-grade dysplasia, which both are markers of increased cancer risk. Differences between LS and sporadic colorectal neoplasms have been extensively studied, demonstrating differences in molecular genetic alterations [
23‐
26] and histopathological characteristics [
6,
15,
27,
28]. The accelerated carcinogenesis in LS does not seem to be reflected by a difference in degree of apoptosis with sporadic carcinogenesis [
29,
30], although several differences in expression of apoptosis-regulating proteins have been found [
30,
31,
32]. To our knowledge, FasL expression has not been studied before in LS-associated neoplasms. Our results indicate similar expression patterns of FasL in LS and sporadic neoplasms. So, FasL expression seems to occur early in colorectal carcinogenesis, irrespective of the sporadic or hereditary origin of the lesions.
We found that FasL expression was more prevalent in carcinomas than in adenomas, consistent with other reports [
10‐
13]. It was recently demonstrated that MSI-H tumours were associated with a reduced frequency of FasL expression relative to non-MSI-H tumours [
33]. In our study, a similar trend was seen although this did not reach statistical significance.
Infiltration of lymphocytes is a prominent feature of MSI-H tumours, including LS-associated colorectal cancer [
21,
22,
27,
34]. This was confirmed in the present report. In this study, we show for the first time that LS-associated adenomas display the same feature. Moreover, we found that the mean number of TILs was higher in LS adenomas with high-grade dysplasia compared with those with low-grade dysplasia. Only few studies are available that have evaluated the degree of infiltration with lymphocytes in adenomas [
35‐
37]. From these studies, it appears that the number of TILs increases in the course of the adenoma-carcinoma sequence, which is corresponding to our results. It has also been shown that the number of TILs is an independent prognostic factor in colorectal cancer [
38‐
40]. As LS-associated colorectal cancer and sporadic MSI-H colorectal cancer are associated with better survival rates than sporadic cases [
1], it has been suggested that the presence of a cytotoxic anti-tumour immune response may at least in part contribute to the survival advantage described in these patients [
34,
38]. Whether TILs in LS-associated and MSI-H sporadic tumours are really activated cytotoxic T-cells remains to be demonstrated.
The aim of our study was to determine whether higher FasL expression in neoplasms was associated with lower levels of tumour cell apoptosis or increased levels of apoptotic TILs, reflected by reduced TIL density. We found no such correlations in LS-associated neoplasms. In sporadic cases, a positive rather than a negative correlation between FasL expression and tumour cell apoptosis was found. This finding may be a consequence of elevated FasL levels triggering tumour cell apoptosis in a manner similar to autocrine suicide of activated T-cells [
41]. Our findings are in contrast with those from Houston et al, who did not find a correlation between FasL expression and degree of tumour cell apoptosis [
42]. It must be noted that their series consisted of only ten colorectal tumours, which may explain the absence of a correlation in their study. The same research group recently found a non-significant negative association between intensity of FasL staining and number of TILs in a series of 91 colon carcinomas [
43]. Two studies reported a positive correlation between FasL expression and apoptosis in TILs [
43,
44], whereas we did not find an association between FasL expression and TIL density. The background for the discrepancy between these data and ours is unclear. A possible explanation may lie in the fact that in the other studies TUNEL and ISNT were used to assess TIL apoptosis. Both these techniques have poor specificity for apoptosis and their use is currently discouraged [
7,
45].
The question whether the Fas counterattack plays a role at all in colorectal carcinogenesis is surrounded with controversy [
46,
47]. The idea behind the concept is that FasL expression by tumour cells may enable those cells to kill anti-tumour immune effector T-cells that infiltrate the tumour. Many tumours and tumour cell lines of varying histologic origin express FasL [
48]. Several functional studies have shown that FasL positive colon tumour cells can induce apoptosis of Fas-sensitive lymphocytes in vitro [
49,
50]. In addition, inhibition of FasL expression reduced tumour development and growth in a xenograft experiment [
51]. There are however also experimental data which seriously question the concept of the Fas counterattack. In a replication study, FasL-expressing colon cancer cells did not induce apoptosis in Fas-sensitive target cells [
52]. In a gene transfer experiment, Fas resistant colon cancer cells, subcutaneously injected into mice, rapidly regressed upon transfection with FasL [
53]. In another experiment, cytotoxic T-cells were not sensitive to FasL displayed on the surface of antigen-presenting cells [
54]. Taken together, the role of the Fas counterattack in colorectal carcinogenesis is at least questionable [
48].
In conclusion, we observed upregulation of FasL during colorectal carcinogenesis, both in LS-associated and in sporadic cases, without a correlation with the number of TILs. Our data do not support an important role for the Fas counterattack as a mechanism behind the difference between colorectal carcinogenesis in LS-associated and sporadic cases.