Nestin is a wide-spectrum abluminal cell marker of salivary gland tumors

Authors

  • Hiroyuki Yanai,

    Corresponding author
    1. Department of Pathology, Okayama University Hospital, Okayama, Japan
    • Correspondence: Hiroyuki Yanai, MD, PhD, Department of Pathology, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. Email: yanaih@md.okayama-u.ac.jp

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  • Yasuharu Sato,

    1. Department of Pathology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
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  • Hitoshi Nagatsuka,

    1. Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
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  • Tadashi Yoshino

    1. Department of Pathology, Okayama University Hospital, Okayama, Japan
    2. Department of Pathology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
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  • Disclosure statement: The authors have no conflicts of interest to declare.

Abstract

Nestin is an intermediate filament that was first identified in neural progenitor cells. It is expressed in various cell types in the nervous system as well as in other systems. In the present study, we investigated nestin expression in non-neoplastic salivary gland tissue and in salivary gland tumors. In non-neoplastic salivary glands, nestin expression was observed in only a few abluminal cells. In contrast, diffuse nestin staining was observed in the abluminal cells of pleomorphic adenoma (11 of 11 cases), basal cell adenoma (7 of 7 cases), and epithelial-myoepithelial carcinoma (2 of 2 cases). The stromal cells in basal cell adenoma also expressed nestin. In adenoid cystic carcinoma (6 of 7 cases) and polymorphous low-grade adenocarcinoma (3 of 3 cases), nestin positive cells were observed focally. Nestin was not detected in Warthin tumor (6 cases), classical acinic cell carcinoma (2 cases), mucoepidermoid carcinoma (5 cases), or salivary duct carcinoma (4 cases). Because the nestin expression pattern in each histological salivary gland tumor type is unique, nestin could be a very useful abluminal cell marker for the diagnosis of salivary gland tumors.

Nestin was first identified as an intermediate filament in neural progenitor cells.[1, 2] Nestin expression was subsequently observed in progenitor cells and adult tissues outside of the central nervous system.[3, 4] Previous immunohistochemical studies showed that nestin expression was also observed in epithelial tumors of the pancreas, prostate, breast, skin appendage, and uterine cervix.[5-12] Recently, nestin has been reported as a putative marker of cancer stem cells in various tumors.[7]

The salivary glands develop various types of tumors. Classification of salivary gland tumors is based on their cellular components and structural characteristics. The cells of salivary gland tumors are divided into luminal and abluminal. Abluminal cells exhibit a wide spectrum of phenotypes; some express myoepithelial markers such as α-smooth muscle actin and calponin, whereas abluminal cells that lack these markers are recognized as basal cells.

Levin et al. studied nestin expression in salivary gland tumors in vitro.[13] However, to the best of our knowledge, there have been no systematic immunohistochemical studies published on nestin expression in salivary gland tumors. In this study, we investigated nestin expression in normal salivary glands and in the major types of salivary gland tumors and examined the usefulness of nestin immunostaining as a diagnostic tool.

Materials and Methods

This study was approved by the research ethics committee of Graduate School of Medicine, Dentistry, and Pharmaceutical Science of Okayama University.

Salivary gland tumors were identified in the surgical pathology files of Okayama University Hospital, from 2004 to 2012. The type and number of tumors are listed in Table 1. All slides were reviewed by the authors (H.Y., Y.S, and H.N.) and diagnoses were confirmed.

Table 1. Tumor types and their nestin expression patterns
Tissue typenNegativeFocalDiffuse
Non-neoplastic salivary glands423480
Warthin tumor6600
Pleomorphic adenoma110011
Basal cell adenoma7007
Acinic cell carcinoma2200
Mucoepidermoid carcinoma5500
Salivary duct carcinoma4400
Epithelial-myoepithelial carcinoma3003
Adenoid cystic carcinoma7160
Polymorphous low-grade adenocarcinoma3030

Immunohistochemical studies were performed using formalin-fixed paraffin-embedded sections. Antibodies against nestin (mouse monoclonal, 10c2, 1:100; Santa Cruz Biotechnology Santa Cruz, CA, USA), p63 (mouse monoclonal, 4A4, prediluted; Nichirei, Tokyo, Japan), α-smooth muscle actin (mouse monoclonal, 1A4, 1:50; Dako, Glostrup, Denmark), and S-100 protein (rabbit polyclonal, 1:500; Dako) were used as the primary antibodies. Immunostaining was performed with an automated stainer (Benchmark XT; Ventana Medical Systems, Tucson, AZ, USA) using the ultraView staining kit (Ventana). For nestin and p63 staining, antigen retrieval was performed in CC1 buffer (Ventana) for 60 min. Normal nerve or endothelium served as an internal positive control.

The nestin staining pattern was classified as diffuse (more than 30% of abluminal cells stained) or focal (30% or less of abluminal cells stained). Non-neoplastic salivary glands surrounding the tumors were also evaluated for nestin expression in the same manner.

Results

The results of nestin immunohistochemistry in salivary gland tissue and tumors are summarized in Table 1. In non-neoplastic salivary gland tissue, nestin was detected in the endothelium and nerve fibers (Fig. 1a). Although most luminal cells and abluminal cells were negative, a few α-smooth muscle actin positive myoepithelial cells of the acini or the intercalated ducts expressed nestin in 8 of 42 salivary glands. (Fig. 1b).

Figure 1.

Nestin immunohistochemistry in normal salivary glands. Nestin is expressed in the capillary endothelium. (a) Although most abluminal cells lack nestin, (b) exceptional expression is observed in myoepithelial calls of some non-neoplastic salivary gland tissues.

In all pleomorphic adenomas (n = 11), the myoepithelial cells in both the ductal structure and mesenchymal-like area showed diffuse nestin staining. These cells were strongly positive for nestin. Myoepithelial cells with plasmacytoid features showed globular pattern positivity for nestin. However, myoepithelial cells showing squamous metaplasia lacked nestin expression. The luminal cells of the neoplastic ducts were negative for nestin. (Fig. 2).

Figure 2.

Nestin immunohistochemistry in pleomorphic adenoma. (a, c, e, and g) H&E stain and (b, d, f, and h) nestin immunohistochemistry. Nestin is diffusely expressed in the myoepithelial cells in (b) the tumor glands and in (d) the stroma-like cells. (f) Inclusion body-like structure in myoepithelial cells with plasmacytoid features are positive for nestin. (h) Squamous epithelium in myoepithelial cells lack nestin expression.

The epithelial cells in the basal cell adenomas (n = 7), which were located beside the stroma, were diffusely positive for nestin. Immunohistochemistry for α-smooth muscle actin and p63 showed that the nestin-positive epithelial cells also expressed these markers. The stromal cells in all cases of basal cell adenoma also expressed nestin. Nestin-positive stromal cells were positive for S-100 protein and negative for α-smooth muscle actin and p63 (Fig. 3).

Figure 3.

Immunohistochemistry of abluminal cell markers in basal cell adenoma. (a) nestin, (b) S-100 protein, (c) α-smooth muscle actin, and (d) p63 staining. Stromal cells express both nestin and S-100 protein.

Focal nestin expression was observed in 5 of 6 adenoid cystic carcinomas. Nestin-positive cells were located at the periphery of tumor cell nests or lining the false lumen and exhibited a characteristic dot-like pattern in some cells (Fig. 4). Nestin-positive cells also positive for myoepithelial cell markers such as α-smooth muscle actin and p63 (Fig. 5). There was no association between histological pattern and nestin expression.

Figure 4.

Immunohistochemistry of adenoid cystic carcinoma. (a, b, and c) nestin, (d) α-smooth muscle actin, (e) S-100 protein, and (f) p63 staining. (a) Nestin expression is observed in the cells at the periphery of the nests or in the cells lining the pseudolumen. In some tumors, nestin immunostain shows dot-like pattern (b arrows). Nestin positive cells also express myoepithelial markers but lack S-100 protein.

Figure 5.

Nestin immunohistochemistry in epithelial-myoepithelial carcinoma. (a) H&E, and (b) nestin. Nestin expression in myoepithelial cells is diffuse.

The myoepithelial cells in the epithelial-myoepithelial carcinoma (n = 3) showed diffuse and strongly positive nestin immunostaining (Fig. 5).

Some tumor cells in the polymorphous low-grade adenocarcinoma (n = 3) were positive for nestin. Nestin-positive cells were also positive for S-100 protein and negative for α-smooth muscle actin and p63 (Fig. 6). There was no relationship between nestin positivity and histological pattern. Some tumor cells in the monolayer tubular structure also expressed nestin.

Figure 6.

Immunohistochemistry of abluminal cell markers in polymorphous low-grade adenocarcinoma. (a and b) nestin, (c) S-100 protein, and (d) α-smooth muscle actin. Nestin expression is found focally. In some tumor cells in single layered tubular structure shows nestin positivity. Positive cells lack myoepithelial marker.

Nestin expression was not detected in Warthin tumors (n = 6), classical acinic cell carcinomas (n = 2), mucoepidermoid carcinomas (n = 5), or salivary duct carcinomas (n = 4).

Discussion

Salivary gland tumors are classified into three major subtypes according to their cellular constitution: mainly luminal cells, mixed luminal and abluminal cells, and predominantly abluminal cells.[14] In the present study, nestin-positive cells were detected in tumors with abluminal cells, i.e. pleomorphic adenomas, basal cell adenomas, epithelial-myoepithelial carcinomas, adenoid cystic carcinomas, and polymorphous low-grade adenocarcinomas.

Each tumor type exhibited a characteristic nestin immunostaining pattern. Nestin expression was diffuse and strong in pleomorphic adenoma, basal cell adenoma, and epithelial-myoepithelial carcinoma. In contrast, in adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma, nestin staining was focal and some cells showed a dot-like pattern. In pleomorphic adenoma, myoepithelial cells showing squamous metaplasia lacked nestin expression. These results suggest that the nestin expression pattern is not uniform in abluminal cells and responsible for differentiation of abluminal cells and characteristic tumor morphogenesis.

Basal cell adenomas showed a unique nestin expression pattern. Nestin was not only detected in some epithelial cells, but also in some stromal spindle cells in basal cell adenomas. Stromal cells in the basal cell adenoma show some characteristic features, such as S-100 protein expression. Dardick et al. thought that the stromal cells in basal cell adenoma were a special type of myoepithelial cells.[15] The findings of the present study may support this hypothesis.

Immunohistochemical staining of some abluminal cell markers such as calponin, α−smooth muscle actin, cytokeratin 14, p63, S-100 protein, and glial fibrillary acidic protein (GFAP) also serve as diagnostic tools. Of these markers, GFAP expression is exceptional in non-neoplastic abluminal cells. Nestin expression pattern in non-neoplastic abluminal cells was similar to GFAP. The present study showed that nestin positivity was observed in neoplastic abluminal cells in characteristic patterns and staining intensity. These findings suggest that nestin expression in salivary gland epithelium support a diagnosis of neoplastic lesion and difference of positivity in abluminal cells can be useful in differential diagnosis of these tumors. It is expected that nestin immunostaining also serve as a diagnostic aid in limited amount of specimen such as aspiration cytology or needle biopsy.

Previous studies showed that nestin was expressed in non-neoplastic myoepithelium and the tumor cells in basal-like ductal carcinoma of the breast.[6] Other studies showed nestin expression in myoepithelial cells of skin appendage tumors.[10, 11] The results of these studies and the present study suggest that myoepithelial cells or basal cells of some organs have a common mechanism of nestin expression.

In melanoma cells, expression of the neural crest transcription factor SOX10 was associated with nestin expression.[16] Cimino-Mathews et al. demonstrated that approximately 70% of basal-like breast carcinomas express SOX10.[17] In a recent immunohistochemical study, Ohtomo et al. showed that most adenoid cystic carcinomas, epithelial-myoepithelial carcinomas, acinic cell carcinomas, and pleomorphic adenomas of the salivary glands were positive for SOX10.[18] According to their study, mucoepidermoid carcinomas, Warthin tumors, and salivary duct carcinomas do not express SOX10 and our study showed that these tumors also lacked nestin. Although non-neoplastic salivary gland tissue and acinic cell carcinoma expressed SOX10, our study revealed that these tissues were negative for nestin. These findings suggest that SOX10 is a key molecule involved in nestin expression in basal-like breast cancer and some salivary gland tumors. However, SOX10 and nestin expression in salivary gland tumors did not necessary overlap. Therefore, we think that there is another molecule that induces nestin expression in salivary gland tumors.

Nestin has been considered a marker of cancer stem cells in tumors of the central nervous system and other organs.[7] However, nestin-positive cells are so numerous that it seems nestin is not a cancer stem cell marker in salivary gland tumors.

In summary, we detected nestin expression in some salivary gland tumors. Since nestin expression in the salivary glands is limited to some types of tumors and each histological type has a characteristic expression pattern and intensity, nestin immunohistochemistry could be useful in the diagnosis of salivary gland tumors. However, the role of nestin in the pathogenesis and morphogenesis of salivary gland tumors and mechanism of nestin expression are subjects for further studies.

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