Original Article
Costimulatory molecule OX40/OX40L expression in ductal carcinoma in situ and invasive ductal carcinoma of breast: An immunohistochemistry-based pilot study

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Abstract

OX40, a membrane-bound member of the tumor-necrosis-factor-receptor (TNFR) superfamily, plays an important role in proliferation, survival and infiltration of activated T cells via binding to OX40L. Recent studies indicate that OX40/OX40L system mediates the adhesion and infiltration of adult T cell leukemia (ATL). Previously, we detected OX40 expression in breast carcinoma cell lines and tissues. The correlation of expression of OX40 and OX40L and clinical features in breast carcinogenesis, however, has not been well characterized. The expression of OX40 and OX40L in 107 invasive ductal carcinomas (IDCa), 9 ductal carcinomas in situ (DCIS), and 31 fibroadenomas from breast tissues and its relationship with the clinical features were determined using immunohistochemistry (peroxidase-conjugated polymer method, ChemMate™ Envision™ Detection kit). The positive immunostaining rates for OX40 in IDCa, DCIS and fibroadenomas from breast tissues were 85.0%, 66.7% and 38.7% respectively, showing a significant difference in OX40 expression among IDCa, DCIS and fibroadenoma of breast (z = 5.206, P = 0.001). Increased staining intensity of OX40 was associated with TNM stages (z = 2.112, P = 0.017). Meanwhile, a relation of OX40 expression with lymph node metastatic status in IDCa was found (P = 0.041). The expression of OX40L did not show any obvious difference among IDCa, DCIS and fibroadenomas from breast tissues. OX40L expression was also not related to histopathological parameters in IDCa except for progesterone receptor (PR) being positive (P = 0.005). However, a high coincidental positive rate for OX40 and OX40L was observed in biopsy samples with IDCa (P = 0.017, Kappa = 0.231). The present results suggest that high OX40 expression may be associated with malignant transformation, progression, invasion and metastasis in breast cancer biology.

Introduction

OX40 (CD134), initially identified as a T-cell activation marker in 1987 [1] and a membrane-bound member of the tumor-necrosis-factor-receptor (TNFR) superfamily, is an important costimulatory molecule during the immune response [2]. The OX40 protein is primarily expressed on the activated CD4+ T cells in rats [2] and on activated CD4+ and CD8+ T cells in mouse and human [4], [5], [6]. OX40L (OX40 ligand), a molecule originally identified as human gp34, is a type-II membrane protein of 34 kD [4], [5]. As a member of the tumor-necrosis-factor (TNF) superfamily, it is expressed mainly on activated APCs, such as activated B cells [4], [5], [6], mature dendritic cells (DCs) [3], activated macrophages, umbilical vein endothelial cells (HUVECs) and human T cell leukemia virus type I (HTLV-1) infected T cell lines [4], [5], and also has been found on activated T cells [7]. In vitro studies have shown that OX40/OX40L represents a costimulatory signal to promote continued proliferation and clonal expansion of CD4+ T cells during the later phase of immune response [3], [8], [9], [10], and to maintain T-cell long-term survival [11]. In addition, the OX40/OX40L pathway has been proven to play a critical role in the adhesion of activated normal CD4+ T cells to endothelial cells [12], [13]. Furthermore, Imura et al. reported that the OX40/OX40L system also directly mediated adhesion and infiltration of adult T cell leukemia (ATL) which expressed OX40 [14], [15].

To further explore the physiological and pathological functions of OX40/OX40L, we previously generated a mouse anti-human OX40 monoclonal antibody (mAb) (clone 1F7) [16] and mouse anti-human OX40L monoclonal antibody (mAb) (clone 9H10) [17]. Using these antibodies, we detected OX40 expression in tumor cell lines of epithelial origin, as well as in breast carcinoma tissues. Breast cancer is the most common cancer in women, and the incidence of breast cancer is increasing in China due to the improvement of people's quality of life and changes in the people's lifestyle. Accumulating data have proved that costimulatory molecules, such as CD40 and B7-H4, participate in tumor progression [18], [19]. Therefore, in order to investigate the significance of OX40 expression in breast carcinoma and the underlying mechanisms, we studied OX40/OX40L in fibroadenomas, ductal carcinoma in situ (DCIS), and invasive ductal carcinomas (IDCa) in an immunohistochemistry-based pilot study, and to identify its role in breast cancer biology. It was found that OX40 expression in breast cancer was associated with tumor development and lymph metastasis, while OX40L expression showed nearly no relationship with breast cancer histopathological parameters. Thus, OX40/OX40L may be involved in the development of breast cancer.

Section snippets

Patients

In this study, we collected 107 cases of IDCa and 9 cases of DCIS, involving 2 cases of pure DCIS and 7 cases which were found around the invasive component. Thirty-one cases of fibroadenomas from breast tissues were used as a control group. All the cases were randomly selected from archival material of the Department of Pathology of the Second Affiliated Hospital of Soochew University, including resected biopsies or mastectomies. All the hematoxylin/eosin (HE)-stained sections were

OX40 and OX40L expression in breast tumor tissues

Histopathologically, all the 107 surgically-resected breast specimens were confirmed as IDCa. Of the 9 cases of DCIS, there were involved 2 cases of pure DCIS and 7 cases which were found around the invasive component. Of the 46 biopsy samples examined, 31 cases were diagnosed as fibroadenoma (Table 1).

The positive immunostaining for OX40 and OX40L was observed in the breast epithelial cells and cancer cells with different rates in the lesions of IDCa, DCIS and fibroadenomas of breast. Fig. 1

Discussion

OX40 is an important costimulatory molecule that has recently attracted considerable attention in angioimmunoblastic T cell lymphoma (ATLD), mycosis fungoides (MF) and adult T cell leukemia/lymphoma (ATLL) [22], [23]. Imura also reported a significantly high OX40 expression in infiltrating ATL cells in skin [15]. ATL is a leukemia/lymphoma of mature T cells with characteristic hematologic features and endemic incidence [24]. One of the frequent manifestations of ATL is infiltration of leukemic

Acknowledgements

This work was supported in part by National Natural Science Foundation of China 30672390, Doctor Project for Basic Research Q4134803 (China), The National Key Base Research Program of China (2007CB512402), and National Natural Science Foundation of China 30500443.

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    These authors contributed equally to this paper.

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