Cancer testis antigens and immunosurveillance in human cutaneous squamous cell and basal cell carcinomas

: PURPOSE: Non melanoma skin cancer (NMSC) is the most common cancer and comprises basal cell (BCC) and squamous cell carcinoma (SCC). The incidence of SCC increases drastically in immunosuppressed individuals, suggesting a critical role of the immune system in controlling SCC. To find an explanation for the selective immunosurveillance of SCC, we investigated the expression of cancer-testis antigens (CTA), MHC class I and the infiltration by immune cells in BCC and SCC. Experimental design: We determined the expression of 23 different CTA in 63 BCC and 40 SCC biopsies of immunocompetent and in 20 biopsies of immunosuppressed SCC patients by RT-PCR and immunohistochemistry. IgG responses to 36 tumor antigens were measured by Western Blotting and ELISA. MHC-I expression and CD8+ T cell infiltration were analyzed by immunohistochemistry in BCC and SCC of immunocompetent and immunosuppressed patients and in imiquimod-treated BCC patients.RESULTS: We found expression of at least one CTA in 81% of BCC and in 40% of SCC. We did not detect CTA-specific serum IgG. Most SCC, but not BCC, expressed MHC-I and were infiltrated with CD8+ cells. Imiquimod-treated BCC expressed MHC-I and were infiltrated by CD8+ T cells.CONCLUSIONS: We propose that immunosurveillance controls SCC, but not BCC, as the latter lacks MHC-I. This fits with the increased incidence of SCC in immunosuppressed individuals and may explain the relatively low prevalence of CT-antigen expression in SCC as a result of CD8+ T cell driven immunoediting. Abstract Purpose: Non melanoma skin cancer (NMSC) is the most common cancer and comprises basal cell (BCC) and squamous cell carcinoma (SCC). The incidence of SCC increases drastically in immunosuppressed individuals, suggesting a critical role of the immune system in controlling SCC. To find an explanation for the selective immunosurveillance of SCC, we investigated the expression of cancer-testis antigens (CTA), MHC class I and the infiltration by immune cells in BCC and SCC. Experimental design: We determined the expression of 23 different CTA in 63 BCC and 40 SCC biopsies of immunocompetent and in 20 biopsies of immunosuppressed SCC patients by RT-PCR and immunohistochemistry. IgG responses to 36 tumor antigens were measured by Western Blotting and ELISA. MHC-I expression and CD8 + T cell infiltration were analyzed by immunohistochemistry in BCC and SCC of immunocompetent and immunosuppressed patients and in imiquimod-treated BCC patients. Results: We found expression of at least one CTA in 81% of BCC and in 40% of SCC. We did not detect CTA-specific serum IgG. Most SCC, but not BCC, expressed MHC-I and were infiltrated with CD8 + cells. Imiquimod-treated BCC expressed MHC-I and were infiltrated by CD8 + T cells. Conclusions: We propose that immunosurveillance controls SCC, but not BCC, as the latter lacks MHC-I. This fits with the increased incidence of SCC in immunosuppressed individuals and may explain the relatively low prevalence of CT-antigen expression in SCC as a result of CD8 + T cell driven immunoediting.


Introduction
Non melanoma skin cancers (NMSC) are the most common cancers in the Caucasian population and include squamous (SCC) and basal cell carcinoma (BCC).
According to the WHO, 2-3 million new cases of NMSC occur globally each year 1 .
NMSC presents a low metastatic potential, however, the tumor can cause substantial local damage if not treated early. The standard surgical care of SCC and BCC often causes severe pain and disfiguration (recently reviewed in (1)).
The risk to develop NMSC significantly increases up to 250 fold in immunosuppressed patients, such as organ transplant recipients (OTR). In addition, aggressive and metastatic variants of SCC develop in OTR, which represent a critical health burden (2,3). The ratio BCC:SCC is 4:1 in immunocompetent patients, but 1:10 or higher in OTR, suggesting a critical role for immunosurveillance in SCC but not in BCC. However, studies explicitly addressing this issue are lacking.
Although NMSCs are frequently infiltrated by immune cells (for review see (4)), the immune system often seems incapable of eradicating the tumor. Downregulation of E-Selectin and recruitment of regulatory T cells (T reg) (5) as well as malfunctioning intratumoral myeloid dendritic cells (6) have been proposed as mechanisms that compromise local tumor-specific immunity in SCC. In the case of BCC, MHC-I absence or down-regulation (7,8) and the presence of T regs (9) was observed.
Immunotherapy is an alternative treatment approach, especially for cancers that can't be surgically removed due to their site or to multiple metastases. Usually, patients are immunized with tumor-associated antigens in order to induce or boost tumorspecific immunity and this approach has shown objective clinical responses in some patients. Many of those vaccines contain tumor-specific differentiation antigens 5 (Melan-A/MART-1, tyrosinase, gp100 in the case of melanoma), overexpressed antigens (e.g. p53, survivin) or cancer-testis (CT) antigens (e.g. NY-ESO-1, members of the MAGE family) (10,11). CT-antigens belong to an extended family of antigens that are expressed by a large variety of malignancies and that are absent from healthy tissue except for testis and placenta. In addition, cancer patients often develop spontaneous immune responses towards CT-antigens, which illustrates their immunogenicity (for review see (12)). In contrast to other malignancies including melanoma, tumor-specific immune responses and tumor-associated antigens that may be the target of such responses are scarcely investigated in NMSC and as a consequence, it is unknown whether immunotherapy may be a suitable therapeutic modality for these cutaneous malignancies. In a recent study, the expression of the CT-antigen MAGE-A4 was identified in cutaneous SCC on an immunohistochemical level (13), but no other data regarding the expression and immunogenicity of CTantigens in BCC and SCC are available.
In this paper we present the first wide screening for CT-antigen expression in SCC and BCC in immunocompetent and -suppressed (OTR) patients. Furthermore, when comparing BCC and SCC with respect to MHC class I expression and infiltration by CD8 + T cells, we found that both immunologically relevant parameters were significantly lower in BCC. This may offer an explanation for the selective increase of SCC-incidence in OTR.
Topical application of imiquimod, a Toll-like receptor (TLR) 7 agonist, results in total regression of the lesion in a large proportion of NMSC patients. Interestingly, we found a significant upregulation of MHC class I expression in BCC upon imiquimod treatment, which adds another possible mode of action to the presumed ones, including stimulation of innate immunity and the induction of apoptosis in tumor cells (14,15 using Ventana reagents for the entire procedure.

Image analysis and quantification
Images of the stained paraffin sections were acquired on a Zeiss Axiophot HAL100 All CT-antigens were more frequently expressed in BCC than in SCC, except for MAGE-A3 (Fig. 1B). In addition, BCC expressed more antigens per biopsy on average than SCC ( Figure 2

No spontaneous humoral immune responses to CT antigens in NMSC patients
CT-antigens often cause spontaneous humoral responses in patients with cutaneous malignancies like melanoma (18,24) and cutaneous T-cell lymphoma (25). We used western blotting and ELISA to investigate whether also NMSC patients display humoral immune responses to tumor antigens.
We screened 34 sera from NMSC patients (9 SCC, 25 BCC) for antibodies against an array of CT-antigens by western blot (data not shown) and ELISA ( Figure S2).
The absence of tumor antigen-specific IgG in sera from patients with NMSC was in contrast with data from most other malignancies and was therefore unexpected. We explain the virtual absence of tumor-specific humoral immune responses by the relatively small tumor load of NMSC in comparison to other tumor entities. It has been well documented, that titers of tumor-specific IgG increase with progressive disease (26,27) i.e. with increasing tumor load and/or the occurrence of metastases, both of which are rare in NMSC.

Low expression of MHC class I and paucity of infiltrating CD8 + cells in BCC suggests limited immunosurveillance
The selective increase of the SCC risk in immunosuppressed individuals suggests a more pronounced role of immunosurveillance in SCC than in BCC. One possible explanation may be the relative absence of suitable tumor-associated antigens in BCC, however, our results show the opposite (Fig 1-2 Fig. 4 and 5), which confirms previous observations (7,8). Both the expression of MHC class I as well as the number of infiltrating CD8 + cells was most prominent in the invasive front of SCC biopsies and statistical analysis revealed a significant correlation between these two parameters (Fig. 5C) Nevertheless, although MHC-I staining does not differ between SCC OTR and SCC patients, the number of CD8 + cells in the intra-, peri-and invasive regions of the tumor are decreased in SCC OTR compared to immunocompetent patients (Fig. 5B).
This coincides with a recent study showing an overall decrease of cytotoxic T cells in OTR (28) and further outlines the role of CD8 + -mediated immune control of SCC and probably not of BCC. These results fit the fact that the activated sonic hedgehog pathway presumably is the driving force in BCC formation (for review see (29)) and that immunological tumor defence therefore has a minor impact on BCC formation.

Local treatment with imiquimod results in upregulation of MHC class I and increased peritumoral CD8 + T cell infiltration in BCC
Repeated topical application of imiquimod, a TLR7-agonist, results in regression of superficial skin cancers, presumably through the induction of innate immunity and local tumor-specific CD8 + T cells, as has been reported for SCC (5,33). As the CD8 + T cells of imiquimod-treated BCC patients remain in the peritumoral region and do not seem to penetrate the BCC nests (Fig. 6), their tumor-antigen specificity and antitumor effector functions remain to be confirmed for BCC.
Taken together, we found that BCC expresses no or low levels of MHC class I molecules, and that -probably as a result thereof -significantly lower numbers of