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. 2010 Feb 10;22(2):89–94. doi: 10.1016/j.sdentj.2010.02.005

A study of HER-2/neu expression and silver binding nucleolar organizer regions (Ag NORs) in perilesional normal salivary gland tissue

Essam Taher MA Gaballah 1,2,⁎
PMCID: PMC3723257  PMID: 23960481

Abstract

Salivary gland neoplasms comprise phenotypically and biologically diverse lesions of uncertain histogenesis.

Objectives

The present study was carried out to assess the immunohistochemical expression of HER-2/neu protein and of silver binding nucleolar organizer regions in normal salivary tissue.

Materials and methods

Twenty paraffin blocks of salivary tumors containing surrounding normal salivary tissue were used in the study. One section was processed to investigate HER-2/neu protein expression and another section was stained to assess the mean Ag NOR counts in normal salivary tissue.

Results

Only two cases of the studied normal salivary tissue showed HER-2/neu expression (10%), which was expressed only in ductal cells with 4.2 ± 2.1 mean number of reactive cells per five high power fields (HPFs). Statistically significant (P = 0.01) higher mean Ag NOR counts in ductal (2 ± 0.83) than in acinar cells (1.2 ± 0.01) of normal salivary tissue were found.

Conclusion

It was concluded from the data of the present study that ductal cells of normal salivary tissue are a proliferative pool and an oncogenic target which plays an important role in the histogenesis of salivary gland tumors.

Keywords: Histogenesis, HER-2/neu expression, AgNORs, Salivary gland neoplasms

1. Introduction

Two main theories exist regarding the histogenesis and development of salivary gland neoplasms. The first one is the multicellular theory in which neoplasms arise from different mature cells that have counterparts within the salivary gland unit. For example, oncocytic tumors are believed to arise from striated duct cells, acinus cell tumors from acinar cells and mixed tumors from intercalated duct cells and myoepithelial cells (Ellis and Auclair, 1996).

The second theory, the semipluripotential bicellular theory, states that basal cells of the excretory and intercalated ducts serve as stem cells or reserve cells for the differentiation of salivary gland units, and that salivary neoplasms arise from either one of these reserve cells. Therefore, Warthin’s tumor, oncocytic tumors, adenoid cystic carcinoma and acinus tumors are thought to arise from intercalated duct reserve cells and mucoepidermoid carcinoma and other more malignant neoplasms are thought to arise from the excretory duct reserve cells (Ellis and Auclair, 1996; Eisele and Johns, 1988).

In addition to the previously mentioned theories, Redman (1995) proposed that acinar cells is the primary candidate as the stem cell of rat parotid gland which can differentiate into other epithelial cells including ducts. A fourth theory of salivary gland tumors histogenesis is that the intercalated duct reserve cells are thought to represent one aspect of the potential bidirectional differentiation to ducts and acini (Eversole, 1971). Finally, other investigators suggested a role of myoepithelial cells in the histogenesis of some salivary gland tumors (Hubner et al., 1971).

HER-2/neu is a cell membrane surface bound receptor tyrosine kinase and is normally involved in the signal transduction pathways leading to cell growth and differentiation. Salivary gland carcinomas were found to show an amplification of the HER-2/neu gene or overexpression of it’s protein product which was associated with increased disease recurrence and worse prognosis (Batsakis et al., 1989; Glisson et al., 2004; Giorgio et al., 2007).

Among the methods of evaluation of the proliferative activity is the argyrophilic nucleolar organizer region (Ag NOR) technique, which uses silver to stain the proteins associated with the active nucleolar organizer regions (NORs). NORs, which transcribe ribosomal RNA (rRNA), are DNA segments found on the short arms of acrocentric chromosomes 13, 14, 15, 21 and 22 of the human species. Active NORs are associated with non-histone and argyrophilic proteins, which are known as Ag NORs when stained with silver. Number and size of Ag NORs change according to the rhythm of ribosomal RNA transcription (Schliephake, 2003).

The present study was carried out to assess the proliferative activity of the cells in perilesional normal salivary tissue by detecting their expression of silver binding nucleolar organizer regions (Ag NORs) and HER-2/neu proteins.

2. Materials and methods

The present study was carried out on 20 archival paraffin blocks of salivary tumors obtained from Cairo National Cancer Institute and from Pathology Department of Mansoura Faculty of Medicine in Egypt. These paraffin blocks were selected after the evaluation of hematoxylin and eosin stained sections by microscopical examination to assess the presence of normal salivary tissue around tumors. The selected normal salivary tissue sample fulfilled the following criteria; (a) not invaded by tumor tissue; (b) not included within the tumor tissue; (c) free of inflammatory, dysplastic or other abnormal changes and finally; (d) of reasonable quantity for histopathological and immunohistochemical studies.

From each paraffin block, two sections each of five microns thickness were cut, the first section was processed to evaluate the immunohistochemical expression of HER-2/neu oncoprotein in normal salivary tissue. The paraffin section was deparafinised, rehydrated in graded alcohols, then treated with blocking reagent for 5 min, washed in phosphate buffer working solution (PBS) for 5 min, after which one drop of monoclonal mouse antibody to HER-2/neu (Dako, Denmark) was then placed on the section (1:80 dilution). Negative controls were obtained by omitting primary antibody replacing it with two to three drops of non-specific serum.

Then slides were incubated overnight in a humidity chamber, two to three drops of streptavidin enzyme were placed on each slide, then several drops of the working color reagent (DAB) were placed on each slide, slides were counterstained with Myer’s hematoxylin, dehydrated and covers were mounted using Canada balsam.

For each of the stained slides mean number of reactive cells to anti-HER-2/neu per five high power fields (HPFs) was calculated.

To assess the proteins associated with nucleolar organizer regions (NORs), the second section from each paraffin block was processed according to the method of Ploton et al. (1986). Sections were dewaxed in xylene (3–5 min) and then rehydrated through ethanols to distilled water. The Ag NOR staining solution was prepared by dissolving the gelatin in 1% aqueous formic acid at a concentration of 2%. This solution was then mixed 1:2 volumes, with 50% aqueous silver nitrate solution, to give the final working solution. This solution was immediately poured over the tissue sections, and left for 60 min at room temperature. The silver solution was then washed-off with distilled water and the sections were dehydrated through graded ethanols to xylene and covers were mounted with Canada balsam.

All the stained slides were examined using X100 oil immersion objective lens at a total magnification of X1000. Ag NOR dots were counted, the mean number of Ag NOR dots from five HPFs in each slide was determined for each type of cells with Ag NORs expression separately (Schliephake, 2003). Ag NOR dots that could not be distinguished from each other were counted as a single dot, following the standardized approach described by Crocker et al. (1989).

Statistical analysis was carried out with SPSS 10 software for data analysis and processing.

3. Results

3.1. Immunohistochemical findings (Table 1)

Table 1.

HER-2/neu expression in the studied normal salivary tissue.

Number of positive cases 2/20
Percentage of positive cases 10%
Mean number of positive cells/five HPFs ± SD 4.2 ± 2.1
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SD = standard deviation.

Only ductal cells were reactive to anti-HER-2/neu protein. The HER-2/neu positive cells revealed yellowish to brown faint cytoplasmic staining which appeared membranous in some cells lying directly adjacent to the cell membrane (Fig. 1) and in other cells, showed a patchy cytoplasmic distribution (Fig. 2). The HER-2/neu positive cells were evident in two (10%) out of the studied 20 normal salivary tissue cases. The mean number of reactive cells per five HPFs was 4.2 ± 2.1.

Figure 1.

Figure 1

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HER-2/neu expression in ductal cells of normal salivary tissue, note the completely negative acini and in the inset, the membranous pattern of expression (anti-HER-2/neu, original magnification ×40).

Figure 2.

Figure 2

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Cytoplasmic expression of HER-2/neu protein in ductal cells of normal salivary tissue (anti-HER-2/neu, original magnification ×400).

3.2. Ag NORs findings (Table 2)

Table 2.

Mean number of Ag NORs in ductal and acinar cells of the studied salivary gland cases.

Ductal cells Acinar cells Total mean Ag NOR count
2 ± 0.83⁎ 1.2 ± 0.01⁎ 1.6 ± 0.5
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⁎

Significant difference, P = 0.01 (Mann–Whitney test).

Ag NORs were evident as black dots in the cell nucleoli which was expressed in both acinar and ductal cells of all the studied salivary tissue cases. In acinar cells, usually one to two large Ag NOR dots were seen (Fig. 3), meanwhile, ductal cells revealed multiple Ag NOR dots with smaller size than those seen in acinar cells (Fig. 4). The total mean Ag NOR count for both ductal and acinar cells per five HPFs of the studied normal salivary tissue cases was 1.6 ± 0.5. The mean Ag NOR counts in ductal cells of normal salivary tissue was 2 ± 0.83 dots/5HPFs (Table 2) which was found to be statistically significantly higher than that of the acinar cells (1.2 ± 0.01, P = 0.01).

Figure 3.

Figure 3

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One or two (arrow) Ag NOR dots in acinar cells of normal salivary tissue (Ag NOR, original magnification ×1000).

Figure 4.

Figure 4

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Multiple Ag NOR dots in ductal cells nucleoli of normal salivary tissue (Ag NOR, original magnification ×1000).

4. Discussion

Salivary gland neoplasms comprise phenotypically and biologically diverse lesions of uncertain histogenesis. The molecular events associated with their development and clinicopathological heterogeneity are yet not fully understood (Maruya et al., 2004).

The findings of the present work of 10% expression of HER-2/neu protein among normal salivary gland tissue were similar to those in the study of Negri et al. (2008), as they reported 25% expression (two cases of eight) of HER-2/neu protein in normal salivary tissue. The higher value of HER-2/neu expression in the later study may be due to the difference of the method used, as they used in situ hybridization method for HER-2/nue protein detection which may be more sensitive than the immunohistochemical method used in the present study. Similarly, Quirke and coworkers (Quirke et al., 1989) detected HER-2/neu in a wide variety of epithelial and non-epithelial tissues of human fetus. Moreover, HER-2/neu was expressed in epithelial cells of normal breast, it was proposed to control proliferation of normal breast cells (Gompel et al., 1996).

Some studies showed complete absence of HER-2/neu expression in salivary gland tumors (Haltas et al., 2008; Hovek-Gebeily et al., 2007), while others reported a high expression of HER-2/neu among salivary gland tumors (Shintani et al., 1995; Ettl et al., 2008; Gaballah and Zedan, 2006).

The expression of HER-2/neu in normal salivary tissue of the present study may be explained in two ways; the first is that the histologically normal salivary tissue surrounding salivary tumors is undergoing tumorigenesis which may be supported by the findings of previous researches in which normal salivary tissue around tumors revealed expression of other types of oncoproteins (Negri et al., 2008; Gaballah, in press).

The other explanation of HER-2/neu expression in normal salivary tissue is that this protein has a role in the growth and differentiation of normal salivary tissue which may be supported by the findings of other investigators, who reported absence of any prognostic significance in salivary gland carcinoma cases which expressed HER-2/neu. It was suggested that HER-2/neu may play a role in cell differentiation in specific stages of development (Gompel et al., 1996; Haltas et al., 2008; Ettl et al., 2008). However, the expression of HER-2/neu in ductal cells of normal salivary tissue of the present study signals that these cells play an important role in histogenesis of salivary gland tumors. A similar findings of ductal expression of several types of oncoproteins in normal salivary tissue was reported in other studies (Gaballah, in press; Kong et al., 1998; Actis et al., 2002). Membranous and cytoplasmic expressions of HER-2/neu protein were reported in other studies (Gaballah and Zedan, 2006; Cho et al., 1999).

In this study mean Ag NOR counts in normal salivary tissue of 1.6 ± 0.5 was similar to those of Behnam et al. (2006) who reported 1.69 ± 0.19 mean number of Ag NORs in twelve normal salivary gland tissue around salivary tumors. Also, mean Ag NOR counts in normal salivary tissue of the present study was similar to that found in chronic sialadenitis (1.67 ± 0.14) and in some benign neoplasms (1.67 ± 0.11) which was lower than that of malignant neoplasms (3.59 ± 0.55) in the study of Adeyemi et al. (2006).

The mean number of Ag NORs in ductal cells among the cases of the present study (2 ± 0.83) was similar to that reported in the study of Vered and colleagues (Vered et al., 2004) (2.2 ± 1.4). On the other hand, the mean Ag NOR counts in acinar cells of normal salivary tissue in their study (2.13 ± 1.1) was nearly similar to that of ductal cells which contradicts the finding of the present work as acinar cells of the normal salivary tissue revealed a significantly lower (P = 0.01) mean Ag NOR counts (1.02 ± 0.01) than that reported for ductal cells. However, Vered et al. (2004) reported a significantly higher Ag NOR profile area per nucleus for ductal cells (1.8 ± 0.5) than for acinar cells of normal salivary tissue (1.2 ± 0.1).

The role of ductal cells as a main source of salivary gland tumors histogenesis which can be suggested from the findings of the present study is further supported by similar findings of studies investigating the histogenesis of different salivary gland tumors, for example, in mucoepidermoid carcinoma (Dardick et al., 1990; Soini et al., 2001), pleomorphic adenoma, Warthin’s tumor (Soini et al., 2001), adenoid cystic carcinoma (Chisholm et al., 1975), and acinic cell carcinoma (Chaudhry et al., 1986).

The evident increased proliferative activity reported for ductal cells as compared to acinar and other types of normal salivary tissue cells in the present study may be explained by the suggestion of Dayan and colleagues (2002) and of Vered et al. (2004), as they stated that the different behaviour pattern between the acinar and ductal cells probably derived from the impact that the chronic inflammatory infiltrate might have effect on the ductal epithelium located adjacent to it, the presence of chronic inflammation is closely associated with the production of reactive oxygen species (ROS) and metabolites within adjacent cells, with an ultimate result of inducing DNA damage (Larsson et al., 2001) which is capable of inducing activation of proto-oncogenes and/or impairment of tumor suppressor genes in neighboring target cells (ductal cells) which ultimately may pave the way for the development of salivary tumors (Vered et al., 2004; Schwartsbard, 2003).

Although some investigators suggested that myoepithelial cells have been speculated to play an important role in the histogenesis of some salivary gland tumors (Hubner et al., 1971; Kazunovri, 2001; Bruce and Wertheimer, 1967; Soini et al., 1999), others denied its role in salivary gland tumor histogenesis due to quite low proliferative activity (Batsakis et al., 1983; Takabashi et al., 2001; Redman, 1994) which supports the findings of the present study of absence of HER-2/neu expression or Ag NOR proteins in myoepithelial cells of normal salivary tissue cases in the present study.

The low proliferative activity of myoepithelial cells may be explained by the ability of these cells to secrete substances which inhibit proliferation, invasion and metastasis (e.g. proteinase inhibitors like maspin and α-1-protease nexin II) which contribute to anti-invasive matrix for myoepithelial rich salivary gland tumors (Sternlicht et al., 1996). There is also some evidence that myoepithelium might inhibit angiogenesis, further contributing to local growth and inhibition of metastasis (Sternlicht and Barsky, 1997; Zarbo, 2002).

Although many studies suggested that all salivary gland cells may be capable of proliferation, including secretory acinar cells (Maruya et al., 2004; Dayan et al., 2002; Shirasuna et al., 2006; Miyazaki et al., 2002), the findings of the present work presented ductal cells as the only highly proliferative compartment in normal salivary tissue. Therefore, whether one labels generative cells within salivary gland duct system as pluripotential, stem, reserve, basal, transitional or undifferentiated, they are considered as a regenerative pool and oncogenic targets in normal salivary tissue which plays an important role in the histogenesis of salivary gland tumors.

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