Immunohistochemical Expression of C-erb-B2 and Vimentin in Adenoid Cystic Carcinoma and Polymorphous Low-Grade Adenocarcinoma: A Comparative Study

Salivary glands are the site of origin of a wide variety of neoplasms. The histopathology of these tumors is considered to be among the most complex and diverse than any organ in the body [1].  Salivary gland neoplasms are also relatively uncommon with an estimated annual incidence in the United States of 2.2 to 2.5 cases per 100,000 people; they constitute only about 2% of all head and neck neoplasms [1]. Nearly 80% of these tumors occur in the parotid glands, 15% in the submandibular glands and the remaining 5% in the sublingual and minor salivary glands. Benign neoplasms make up about 80% of parotid tumors, 50% of submandibular tumors and less than 40% of sublingual and minor salivary gland tumors. Salivary gland neoplasms are composed of diverse entities of uncertain histogenesis and unpredictable clinical behavior [2-3]. Adenoid cystic carcinoma (ACC) is the second most common salivary gland malignancy, but is the most common in the submandibular, sublingual and minor salivary glands. It occurs equally in men and women, peaks in the 5th decade of life and is more common in Caucasians [2]. Polymorphous low-grade adenocarcinoma (PLGA) of the salivary glands is rare but aggressive tumor. These tumors tend to clinically present in patients over 40 years of age and occur with nearly equal frequency in men and women.  About half of these tumors present in the parotid glands, the minor salivary glands, particularly the palate; lip and tongue are the next most commonly affected sites [2]. The HER-2/neu oncogene is a member of the epidermal growth factor receptor or erb gene family [4] and has an intrinsic tyrosine kinase activity. HER-2/neu was originally identified as a transforming oncogene in chemically induced rat neuroglioblastomas, where a single point mutation in the transmembrane domain of the molecule is sufficient to confer oncogenic activation [5]. Vimentin is a member of the intermediate filament family of proteins. Vimentin plays a significant role in supporting and anchoring the position of the organelles in the cytosol. It provides cells with resilience, absent from the microtubule or actin filament networks, when under mechanical stress in vivo. Therefore, in general, it is accepted that vimentin is the cytoskeleton component responsible for maintaining cell integrity [6-7]. In the present study, we explored the expression of C-erb-B2 and vimentin in ACC and PLGA by immunohistochemical means and sought whether the staining pattern has any association with histological or clinical characters.

Specimens: Thirty consecutive tissue samples (15 ACC and 15 PLGA), histologically diagnosed as epithelial salivary gland tumors were retrieved from the archives of the Oral Pathology Department, College of Dentistry, University of Baghdad. Samples used in the study were preserved as formalin fixed paraffin embedded tissue blocks. In each block, one representative section was stained with hematoxylin and eosin for reassessment of histopathological diagnosis and two other sections were prepared on adhesive slides for detection of vimentin and C-erb-B2 by using immunohistochemistry. Normal salivary glands tissue specimens were obtained from five blocks fixed in 10% formalin and processed to paraffin embedded blocks. Breast adenocarcinoma tissue sections from the teaching laboratories were used as a positive control for both vimentin and C-erb-B2 immunostaining [8]. The negative control slides were prepared from test tissue processed in a manner identical to the test section while omitting the primary antibody and instead adding 20ml of phosphate buffer saline.

Slide preparation: Paraffin blocks were cut in 4 microns thick sections for optimum resolution with staining, and then the sections were carried on adhesive slides and left to dry upright in order to facilitate adhesion between the sections and the charged glass surface. The sections were placed on slides as flat and wrinkle free as possible to optimize stain contact with the tissue. The tissue sections were dried on the slides by heating (overnight at 65°C) in a hot air oven to ensure that any moisture trapped under the tissue is completely eliminated by melting the paraffin and evaporation of the water droplets. The tissue sections were de-paraffinized by dipping into two changes of xylene for 5 minutes at room temperature (xylene  jar  was placed in the oven  few minutes before dipping  the slides into it  in order to enhance  the de-waxing effect). The tissue sections were rehydrated on slides in order to return water to the tissue by dipping slides into ethanol in the following sequential manner:                                              

• Absolute ethanol for 5 minutes.
• 95% ethanol for 5 minutes.
• 70% ethanol for 5 minutes.
• 30% ethanol for 5 minutes.
• Distilled water for 2 minutes.

Antigen retrieval: Formalin or other aldehyde fixation forms protein cross–links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. For the studied antibodies, staining of  formalin fixed tissues required pretreatment with citric buffer PH 8.0 in order to break the protein  cross-links, therefore unmasking the antigens and epitopes in formalin fixed–paraffin embedded  tissue sections, thus enhancing  staining  intensity of the studied antibodies. The slides were put in koplin jar containing antigen retrieval solution and then the slides in the jar were placed in water bath at 98°C for 10 minutes, then the slides were removed from it, left to cool at room temperature for 20 minutes and placed in distilled water for 5 minutes at room temperature.

Evaluation of immunohistochemical staining: The evaluation of C-erb-B2 immunohistochemical expression was based on a 0-3+ scale as follows:

• 0, staining in < 10% of tumor cells or no staining.
• 1+, faint and partial membrane staining in ≥ 10% of tumor cells.
• 2+, weak to moderate complete membrane staining in ≥ 10% of tumor cells.
• 3+, moderate to strong complete membrane staining in ≥ 10% of tumor cells.

Vimentin expression was determined by evaluating the entire available tumoral tissue in the sections under study. Next, it was determined whether the relative number of positive neoplastic cells was inferior (negative cases) or superior (positive cases) to 10% from all of the neoplastic cells in the histological sections evaluated [9]. The positive cases then were scored into three scores according to degree of positivity:

• 1+ > 10% of cells positive
• 2+ 10- 25% of cells positive
• 3+ > 50% of cells positive

Diffuse membranous staining for C-erb-B2 was noted in both malignant tumors while normal salivary gland tissues were not stained with this marker. In breast cancer specimens, C-erb-B2 expression was observed. In PLGA, 2 (13.3%) sections with weak positive expression, 7  (46.7%) sections with moderate positive expression, 5 (33.3%) sections with strong positive expression and only one (6.7%) section with negative expression (Figure 2). Among vimentin positive ACC specimens, six were of cribriform type; seven were tubular type and two specimens were solid type. We found a non-significant positive correlation between vimentin expression scores and tumor grade. Six vimentin positive specimens were observed in low grade or well-differentiated PLGA, four in moderately differentiated and five cases in poorly differentiated tumors with non-significant correlation.

This study shows that both malignant tumors (ACC and PLGA) show a significantly greater expression of C-erb-B2 (p<0.001) compared to controls. This supports the hypothesis that C-erb- B2 expression may confer oncogenic properties to these malignant tumors.

However, within the tumor samples, the correlation of positive staining (weak, moderate and strong) with histological grading was non-significant. ACC is the most common malignant tumor of minor salivary glands, submandibular and sublingual glands. It has a peak incidence in the fourth to sixth decades of life, which is in accordance with what we found (Table 1), [10, 11]. The mean patient age in our study was 58 years which is consistent with previous reports [1]. The association of oral cancer development with aging could be explained by the prolonged exposure to environmental carcinogenesis such as radiation, viruses and chemicals which play as prompting factors in cancer development. The res ults of the present study concerning gender distribution come in accordance with some previous reports [12] but in contrast to others [13]. We found that males were more affected with these malignant salivary gland tumors than females (Table 1). Similar findings were reported from other parts of the world, including Iraq, which found that 55% of patients with malignant salivary gland tumors were males [14, 15]. In contrast to this, other studies did not find a gender predisposition [1]. This difference may be attributed to the small sample size of this study and the higher frequency of smoking in males than females. Another possible explanation for such discrepancy is due to the variation in the method of data collection and the types of the samples used in different studies. The distribution of ACC and PLGA lesions according to the site revealed that the palate was the commonest site affected with these malignant tumors. This finding was in agreement with previous studies that the effect of smoking habit especially the pipe smoking generates more heat on the palate than other forms of smoking and produces a pronounced palatal keratosis or reverse smoker’s palate, which has a significant potential to develop into dysplasia or carcinoma. Our results revealed positive C-erb-B2 expression in all cases of ACC with strong positivity in 26.7% of cases, and in 93.3% of PLGA cases with 33.3% of the cases showing strong positive expression; these results are also consistent with previous reports [16, 17, 18].

In conclusion, we did not find any significant association between C-erb-B2 and vimentin staining and the histological characteristics of the tumors. Further studies should identify other immunohistochemical markers, which are predictive of grading and staging.

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