Abstract

Cancer immunotherapy has highlighted the clinical relevance of enhancing anti-tumor response of CD8+ T cells in several cancer types. Little is known, however, about the involvement of the immune system in extramammary Paget’s disease (EMPD). We exam- ined the cytotoxicity and the effector functions of CD8+ T cells using paired samples of peripheral blood and tumors by flow cytometry. Expression levels of perforin, granzyme B, IFN-g, TNF-a, and IL-2 in CD8+ tumor-infiltrating lymphocytes (TILs) were significantly lower than those in CD8+ T cells of peripheral blood. Significantly higher expression of PD- 1 was found in CD8+TILs than in CD8+ T cells of peripheral blood. A high number of CD8+ cells was significantly associated with poor overall survival (OS) adjusted with age, sex, and clinical stage (hazard ratio [HR] = 5.03, P = 0.045, 95% confidence interval [CI] 1.03– 24.4). On the other hand, the number of PD-1+ cells was not associated with OS or dis- ease-free survival (DFS). Moreover, we found that tumor cells produced immunosuppres- sive molecule indoleamine 2,3-dyoxygenae (IDO). In conclusion, CD8+ TILs displayed an exhausted phenotype in EMPD. IDO expression seemed more relevant in inducing CD8 exhaustion than PD-1 upregulation or PD-L1 expression by immune cells. Restoring the effector functions of CD8+ TILs could be an effective treatment strategy for advanced EMPD.

Introduction

Extramammary Paget’s disease (EMPD) is a rare skin cancer that occurs predominantly in areas with abundant apocrine sweat glands including the axillary, perianal and genital regions [1]. EMPD usually presents as slow-growing carcinoma in situ with a favorable prognosis. However, some EMPD tumors show invasive / metastatic progression and the prognosis is dis- mal in such cases. Five-year survival rate is 84% in patients without metastasis, whereas only 7% in patients with distant metastasis [2]. Standard therapies for advanced EMPD are lacking, and they are often refractory to systemic therapies [3].

Cancer immunotherapy has highlighted the importance of tumor immunity. The presence of tumor-infiltrating lymphocytes (TILs) is essential for anti-tumor immune response. A high number of CD8+ TILs is associated with favorable prognosis, and a high number of tumor- infiltrating regulatory T cells (Tregs) is associated with poor prognosis in several cancer types [4,5]. The capacity of TILs to act as effector cells is hindered by the tumor microenvironment. For example, programmed death-1 (PD-1) is an immuno-inhibitory receptor expressed by lymphocytes that inhibits their proliferation and effector functions after it binds with pro- grammed death ligand-1 (PD-L1). PD-1 upregulation on CD8+ TILs is associated with exhaustion in several cancer types [6–8]. Therefore, the expression of PD-1 or PD-L1 is associ- ated with poor prognosis in various cancer types [9,10]. Therapeutic PD-1 blockade improved overall survival (OS) by enhancing tumor immunity [11,12]. Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-metabolizing enzyme that is upregulated on tumor cells and contributes to the suppression of T cell response in several cancer types [13–15]. Combination therapy with an IDO-1 inhibitor plus checkpoint inhibitors in patients with several cancer types is being tested in a clinical trial [16]. The mechanisms of immune evasion in the tumor microen- vironment have been revealed in many cancer types. However, little is known about the involvement of the immune system in EMPD.

In this study, we examined the cytotoxicity, the effector functions, and PD-1 expression of CD8+ TILs in EMPD by flow cytometry. We also evaluated the association of CD8+ cells and PD-1+ cells with prognosis. In addition, we investigated the tumor-derived immunosuppres- sive molecule IDO by immunohistochemistry (IHC) and quantitative polymerase chain reac- tion analysis (qPCR).