Elsevier

Clinical Immunology

Volume 141, Issue 3, December 2011, Pages 338-347
Clinical Immunology

The comprehensive assessment of local immune status of ovarian cancer by the clustering of multiple immune factors

https://doi.org/10.1016/j.clim.2011.08.013Get rights and content

Abstract

The aim of this study was to evaluate the local immune status of human ovarian cancers by the comprehensive analysis of tumor-infiltrating immune cells and immunosuppressive factors, and to elucidate the local immunity in clinical course. The numbers of CD1α+, CD4+, CD8+, CD57+, forkhead box P3+ and programmed cell death-1+ cells were counted, and the intensity of immunosuppressive factors, such as programmed cell death-1 ligand (PD-L)1, PD-L2, cyclooxygenase (COX)-1, COX-2 and transforming growth factor β1, were evaluated in 70 ovarian cancer specimens stained by immunohistochemistry. Then hierarchical clustering of these parameters showed the four clusters into ovarian cancer cases. Cluster 1, which had significantly better prognosis than the others, was characterized by high infiltration of CD4+ and CD8+ cells. In conclusion the comprehensive analysis of local immune status led to subdivide ovarian cancers into groups with better or worse prognoses and may guide precise understanding of the local immunity.

Highlights

► We found novel approach to explore local cancer immunity by hierarchical clustering. ► Clustering by scoring immune cells and immune suppressive factors made 4 groups. ► Immune cell infiltration and low immunosuppressors were related to good prognosis. ► This study leads to a precise understanding of the local immune status of cancer. ► We provide a salient tool for the application of immune therapies in ovarian cancer.

Introduction

Ovarian cancer is the most lethal gynecologic cancer in the world with > 200,000 patients diagnosed every year and over a half of them dying annually. These deaths are partly due to the fact that more than half of the patients with ovarian cancer are diagnosed at advanced tumor stages (stage III or IV). Although platinum or taxane-based chemotherapies are effective in the treatment of the majority of ovarian cancer cases, most of the patients suffer from recurrence and eventually develop chemo-resistance. Considering the high mortality rate of ovarian cancer due to the absence of curative treatment in the advanced stage or at recurrence, new therapeutic modalities other than chemotherapy and surgery are urgently needed [1], [2], [3].

Tumor immune therapy has long been considered as an alternative modality in the treatment of solid tumors including ovarian cancer. Nevertheless, there have been few reports on clinically successful immune therapies. The failure in immune therapies in such clinical trials is partly ascribed to the phenomenon designated as “tumor immune escape”. It is increasingly understood that the dynamic interaction between tumor cells and immune cells in the local microenvironment plays a pivotal role in cancer development and progression [4]. In the case of advanced cancers, tumor cells establish an immunosuppressive environment regionally and make it difficult to induce immune activation to eliminate cancer cells. In this situation, adoptive immunotherapy, such as a tumor vaccine, is not sufficient to eradicate tumors [5], [6].

The differences in the phenotypes or populations of tumor-infiltrating immune cells, such as CD4+ (helper) T cells, CD8+ (cytotoxic) T cells, CD57+ (NK) cells and CD11c+ (dendritic) cells, have been shown to be associated with different clinical outcomes of solid tumors including colorectal cancer [7], breast cancer [8], gastric cancer [9], [10], lung cancer [11], [12], hepatic cancer [13], [14], melanoma [15], kidney cancer [16] and uterine cervical cancer [17]. In ovarian cancer, several recent studies have shown an association between tumor-infiltrating immune cells and clinical outcomes [18], [19]. We also reported that CD8+ T cell infiltration [20] and NK cell infiltration [21] are associated with a favorable prognosis in the ovarian cancer patient. On the other hand, regulatory T cells, most specific marker of which is FOXP3, in the tumor site play a suppressive role in the local tumor immunity, leading to tumor progression [22], [23].

With respect to the tumor, there are also a wide variety of mechanisms that enable tumor cells to evade an immune attack. These mechanisms include a loss of MHC [24], the upregulation of immunosuppressive factors, such as transforming growth factor β (TGFβ) [25], IL-10, indoleamine 2,3-dioxygenase (IDO) [26] and cyclooxygenases (COX-1 and COX-2) [27] or upregulating negative regulatory signals, such as programmed cell death-1 (PD-1) ligands (PD-L1, PD-L2) and cytotoxic T lymphocyte antigen-4 (CTLA-4) [28], [29], [30], [31]. We reported that PD-L1 expression in ovarian cancer is inversely correlated with tumor-infiltrating CD8+ T cells and is associated with a poor prognosis of the patient [20]. The expression of the immune suppressive factors COX and UL-16 binding protein 2 is also inversely associated with CD8+ T cell infiltration and the prognosis of the patient with ovarian cancer [21], [32]. Thus, there are a variety of reports that suggest that a certain immunosuppressive factor influences the local tumor immunity. However, there are few comprehensive analyses that integrate various immune factors and evaluate the immune status as a whole.

Therefore, in this study, we attempted to explore the status of local immunity in ovarian cancers by integrating various immune parameters presented by the immunohistochemical analysis of clinical specimens. For this purpose, we employed bioinformatics analyses, such as hierarchical clustering, that allows the comprehensive assessment of multiple factors and enables us to determine the relationships among them.

Section snippets

Patients and Samples

Formalin-fixed, paraffin-embedded specimens were obtained from 70 patients who underwent primary surgery for epithelial ovarian cancer at the Kyoto University Hospital. After surgery, all patients received platinum- and paclitaxel-based chemotherapy. The average age of the patients was 55 years old (range, 26–78; standard deviation [SD], 11). At the end of the study, 29 (41%) patients had died from their disease, and 41 (59%) patients were alive. The mean follow-up period was 5 years (range,

Expression of immune-suppressive factors in ovarian cancer specimens and patient prognosis

Immunohistochemical expression of TGFβ1, PD-L1, PD-L2, COX-1 and COX-2 was evaluated in 70 ovarian cancer tissues (Fig. 1). High expression (score 2 or 3) of TGFβ1 was observed in 22 cases (31.4%) and low expression (scored 0 or 1) was observed in 48 cases (68.6%). There was no correlation between the expression of these factors and clinicopathological characteristics such as age, histological type, FIGO stage, TNM classification, and residual tumor state (Supplementary Table 2) [20], [32].

The

Discussion

Recent studies have shown that local tumor immunity is closely associated with clinical course of cancer patient, and several immunological factors, including CD8 T cell count shown in our previous study, serve as prognostic indicator. However, these analyses were mainly done using single factor or combination of several factors, and there are few papers which tried to clarify the immunological background by analyzing multiple immune factors simultaneously. The application of hierarchical

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgments

We are grateful to Mrs. Yuko Hosoe and Ms. Maki Kurokawa for technical assistance. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References (42)

  • D.O. Croci et al.

    Dynamic cross-talk between tumor and immune cells in orchestrating the immunosuppressive network at the tumor microenvironment

    Cancer Immunol. Immunother.

    (2007)
  • S.A. Rosenberg et al.

    Cancer immunotherapy: moving beyond current vaccines

    Nat. Med.

    (2004)
  • R. Kim et al.

    Cancer immunoediting from immune surveillance to immune escape

    Immunology

    (2007)
  • J. Galon et al.

    Type, density, and location of immune cells within human colorectal tumors predict clinical outcome

    Science

    (2006)
  • S. Ishigami et al.

    Prognostic value of intratumoral natural killer cells in gastric carcinoma

    Cancer

    (2000)
  • K.I. Al-Shibli et al.

    Prognostic effect of epithelial and stromal lymphocyte infiltration in non-small cell lung cancer

    Clin. Cancer Res.

    (2008)
  • Q. Gao et al.

    Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection

    J. Clin. Oncol.

    (2007)
  • A. Ladanyi et al.

    Density of DC-LAMP(+) mature dendritic cells in combination with activated T lymphocytes infiltrating primary cutaneous melanoma is a strong independent prognostic factor

    Cancer Immunol. Immunother.

    (2007)
  • J.S. Schleypen et al.

    Cytotoxic markers and frequency predict functional capacity of natural killer cells infiltrating renal cell carcinoma

    Clin. Cancer Res.

    (2006)
  • S.J. Piersma et al.

    High number of intraepithelial CD8+ tumor-infiltrating lymphocytes is associated with the absence of lymph node metastases in patients with large early-stage cervical cancer

    Cancer Res.

    (2007)
  • L. Zhang et al.

    Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer

    N. Engl. J. Med.

    (2003)
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