Adenoid cystic carcinoma (ACC) and polymorphous low-grade adenocarcinoma (PLGA) are salivary gland malignancies with some overlapping histomorphologic features and immunophenotypic profiles, yet their biologic behavior is significantly different. Some histopathologic similarity is expected as both tumors are composed of ductal and abluminal myoepithelial differentiated cells. Both tumors have a marked propensity to infiltrate around nerves. The distinction between ACC and PLGA is important because ACC is clinically more aggressive and infiltrative, requiring a more radical surgical approach. Although these tumors are often readily diagnosed, occasionally differentiation may pose a diagnostic challenge especially when examining tissue from small incision or fragmented biopsies [
Studies [
Although overexpression of both proteins is well documented in many body tissues and tumor types expression in salivary gland neoplasia is limited, and investigations in the use of c-kit in ACC and PLGA have yielded incongruous results [
29 cases of ACC (Figure
Staining patterns in ACC and PLGA. ACC showing a focal cribriform growth pattern as well as areas of streaming mimicking a PLGA (a) (H&E, ×20) and a PLGA with biphasic structures resembling ACC (b) (H&E, ×40). Marked (>60%) granular cytoplasmic bcl-2 protein expression in the cribriform variant of ACC (c) and the tubular type of PLGA (d) (bcl-2, ×40). Intense c-kit membranous and cytoplasmic immunopositivity (>60%) of the luminal cells in ACC (e); weak (<30%) cytoplasmic and membranous staining in PLGA (f) (c-kit, ×40).
Immunohistochemistry was performed on deparaffinized 4
The sections were immersed in 3% hydrogen peroxide in distilled water for 5 minutes and rinsed in TBS pH 7.6 with 0.1% Tween 20 (Sigma, St. Louis, MO). Specimens were incubated with the primary antibodies using the following dilutions: 1 : 50 for bcl-2 and 1 : 100 for c-kit. Chromogen was applied for 5 minutes, and the color developed with diaminobenzidine hydrochloride (DAB, Sigma, St. Louis, MO) resulting in a brown reaction product. Sections were then lightly counterstained with H&E for 1 minute. Appropriate positive controls, tonsil for bcl-2 and skin for c-kit, were used, along with the omission of the primary antibodies as negative controls.
Dense brown nuclear and cytoplasmic staining of the tumor cells was regarded as positive for each antibody. Both membranous and cytoplasmic c-kit staining patterns were included in the analyses. As reported previously [
The clinical and histological features of both ACC (Figure
Clinical findings in ACC and PLGA.
ACC | PLGA | |||
Total number of cases | 29 | 22 | ||
% | % | |||
Primary | 26 | 89.7 | 22 | 100 |
Recurrent | 3 | 10.3 | 0 | 0 |
Site of involvement | ||||
Major glands | 11 | 37.9 | 0 | 0 |
Parotid | 3 | 10.3 | ||
Submandibular | 8 | 27.6 | ||
Minor glands | 17 | 58.6 | 22 | 100 |
Palate | 10 | 34.5 | 15 | 68.2 |
Maxilla | 4 | 13.8 | 2 | 9.1 |
Buccal mucosa | 1 | 3.4 | 2 | 9.1 |
Floor of mouth | 1 | 3.4 | 0 | 0 |
Tongue | 1 | 3.4 | 1 | 4.5 |
Upper lip | 0 | 0 | 2 | 9.1 |
Unknown | 1 | 3.4 | ||
Major : minor glands | 1 : 1.6 | |||
Gender | ||||
Male | 7 | 24.1 | 8 | 36.4 |
Female | 22 | 75.9 | 14 | 63.6 |
Male : female | 1 : 3.1 | 1 : 1.8 | ||
Age in years | ||||
Mean | 46.6 | 57.8 | ||
Median | 43 | 58 | ||
Range | 22–74 | 23–77 | ||
Race | ||||
Black | 27 | 93.1 | 19 | 86.4 |
White | 1 | 3.4 | 3 | 13.6 |
Indian | 1 | 3.4 | 0 | 0 |
Statistical analysis of the inter- and intraobserver readings clearly established the reliability and reproducibility of the assessment of immunostaining of both antibodies used and of the data obtained. The results of the paired sample
Immunoexpression of bcl-2 and c-kit is summarized in Table
Total immunoreactivity of bcl-2 and c-kit observed in ACC and PLGA expressed as number of cases (
Total | ACC | PLGA | ||||||||||
29 | 23 | |||||||||||
% immunostaining | <30 | 30–60 | >60 | <30 | 30–60 | >60% | ||||||
% | % | % | % | % | % | |||||||
bcl-2 | 6 | 20.7 | 2 | 6.9 | 21 | 72.4 | 4 | 17.4 | 6 | 26.1 | 13 | 56.5 |
c-kit polyclonal | 13 | 44.8 | 8 | 27.6 | 8 | 27.6 | 19 | 82.6 | 2 | 8.7 | 2 | 8.7 |
c-kit monoclonal (pilot study) | 26 | 89.7 | 1 | 3.4 | 2 | 6.9 | 22 | 95.7 | 0 | 0 | 1 | 4.3 |
The results of the pilot study with c-kit monoclonal antibody showed moderate to intense positivity in 10.3% of ACC and 4.3% of PLGA, with no significant difference in staining expression or intensity between the tumors. This was different to the c-kit polyclonal antibody which was expressed in 55.2% of ACC and in 17.4% of PLGA. The expression of c-kit staining in both ACC and PLGA was decreased in comparison to c-kit (polyclonal) staining.
The overall distribution of different staining intensities with bcl-2 and c-kit in both ACC and PLGA, analyzed by chi-squared testing, showed a small yet statistically significantly increased moderate to intense staining in ACC, with weaker staining in PLGA (
A number of studies have attempted to differentiate ACC from PLGA via the use of antibodies to GFAP, vimentin, S-100 protein, Ki-67 (MIB-1),
This study demonstrated a statistically significant decrease in staining intensity with c-kit in PLGA when compared to ACC. Although many studies have reported c-kit to be a useful ancillary marker in the diagnosis of ACC and in distinguishing ACC from PLGA, the results of detectable c-kit expression in salivary gland neoplasia remain ambiguous [
We referred previously to a pilot study in which it was determined that the 2 types of c-kit (monoclonal and polyclonal) resulted in dissimilar staining. The discrepancies in the specificity of c-kit staining are reported to be due to the variability of the primary antibodies selected and the influence of factors such as differences in immunohistochemical protocols (including deparaffination, epitope retrieval methods, dilutions, detection reagents used, and immunohistochemical methods), the varying methods of evaluating immunoreactivity, and the limited number of cases reviewed [
This study, unlike previous reports, showed no preference in c-kit expression for major or minor salivary glands in ACC [
Like previous reports, the pattern and intensity of c-kit immunostaining in ACC in this study varied with the different histological subtypes, with the more aggressive tumors showing greater staining intensity [
Whilst the cribriform pattern and mixed subtypes exhibited higher c-kit reactivity than the tubular pattern alone, others found c-kit expression to be higher among solid and tubular subtypes than the cribriform subtype [
There was no statistically significant difference in bcl-2 expression between ACC and PLGA. ACC showed more intense, granular cytoplasmic and/or nuclear bcl-2 reactivity, with PLGAs exhibiting a weak to moderate cytoplasmic and membranous stain. Adjacent salivary gland ducts, lymphatic tissue, and nerves also expressed bcl-2. In the nonneoplastic salivary gland tissue, bcl-2 positivity was observed in ductal but not in acinar cells. Others have also reported bcl-2 expression in basal cells of striated and excretory ducts, indicating that these cells are reserve cells and that acinar, myoepithelial, and most luminal cells are negative for bcl-2 [
Whilst the expression of bcl-2 has been assessed in some salivary gland tumors, namely, pleomorphic adenoma, monomorphic adenoma, Warthin’s tumor, basal cell adenoma, mucoepidermoid carcinoma, adenocarcinoma (NOS), acinic cell carcinoma, adenoid cystic carcinoma, anaplastic carcinoma, squamous cell carcinoma, and basal cell adenocarcinoma, its use as an immunohistochemical marker in the differentiation between ACC and PLGA has not been reported [
A higher expression of bcl-2 was noted in the solid and cribriform types of ACC suggesting that bcl-2 expression might be associated with myoepithelial cells as these types have more myoepithelial than ductal cells [
The distinction between ACC and PLGA is important since the clinical course and prognostic significance differ. It is imperative that immunohistochemical stains used as adjuncts in histological-based diagnosis be simple, accurate, reliable, and reproducible. Whilst use of antibodies such as
C-kit activity may be necessary for tumor maintenance in ACC. However, ACC seems to proliferate despite inactivation of the c-kit signaling pathway; thus selective c-kit inhibition is probably not useful in ACC therapeutic development [
Neither bcl-2 nor c-kit appears to be valuable as single or joint immunohistochemical markers in the diagnostic differentiation of ACC from PLGA.
The authors declare that there is no conflict of interests.