Background
A basal cell adenoma (BCA) is a benign salivary gland basal cell neoplasm (BCN) that shows a dual-cell proliferation of inner luminal/ductal cells and outer abluminal/ myoepithelial or basal cells [
1]. It has tubular, solid, nested, trabecular, and membranous growth with nuclear palisading [
1]. Currently, catenin beta-1 (
CTNNB1) gene mutations are exclusively found in BCA [
2]. The nuclear expression of β-catenin is considered a characteristic feature of BCA because other salivary gland tumors rarely show nuclear staining [
2]. In the presence of Wnt signal stimulation or an activating CTNNB1 gene mutation, β-catenin stabilizes and moves into the nucleus and interacts with T-cell factor (TCF)/lymphoid enhancer factor (LEF), which represents the “on” state of the Wnt/β-catenin signaling pathway and leads to an increase in the transcription of the target gene associated with the pathway [
3].
A basal cell adenocarcinoma (BCAC) is a malignant salivary gland BCN that possesses dual-cell proliferation similar to BCA [
4]. Many authors differentiate BCAC from BCA by their malignant morphological features, such as invasion into surrounding tissues and perineural and angiolymphatic invasion [
4‐
8]. Although BCAC is recognized as the malignant counterpart of BCA, it is difficult to distinguish BCAC from BCA in surgical pathology practice. Several studies have attempted to identify characteristic immunohistochemical markers for the diagnosis of BCAC [
5‐
8]. In other aspects, BCAC is generally recognized to have “low-grade malignancy” in terms of good overall survival [
9]; however, high-grade cases of BCAC (9%), some frequent recurrent cases (16.7–50%), and some fatal cases (2–5.6%) presented by several authors indicate the necessity for differentiating between BCA and BCAC [
5,
7,
9,
10]. These findings suggest that there is some heterogeneity in biological behavior among BCAC cases.
Transducin-like enhancer of split 1 (TLE1) is a member of the Groucho (ortholog of
Drosophila)/TLE gene family located at chromosome 9q21.32 and acts as a transcriptional corepressor [
11]. The association of TLE1 and tumors was first detected in synovial sarcomas through gene expression profiling, and TLE1 was found to be profoundly involved in the Wnt/β-catenin signaling pathway in synovial sarcomas [
12,
13]. Furthermore, TLE1 expression was associated with better prognosis in gastric cancer and human epidermal growth-factor receptor 2 (HER2)-positive or triple-negative breast cancer, and the clinical significance of the expression profile of TLE1 in several carcinomas was recently reported [
11,
14]. To the best of our knowledge, there are no relevant reports regarding TLE1 expression in salivary gland neoplasms.
During the absence of the Wnt signal and impeded migration of β-catenin to the nucleus, TLE interacts with TCF/LEF and histone deacetylase (HDAC) instead of β-catenin and functions to repress the transcription of Wnt target genes, representing the “off” state of the Wnt/β-catenin signaling pathway [
15]. We hypothesized that if the dysregulation of the Wnt/β-catenin signaling pathway could be attributed to the tumorigenesis of BCA/BCAC, there might be differences in TLE1 expression between BCA and BCAC. This is the first report on TLE1 expression from the perspective of Wnt/β-catenin signaling. Therefore, we performed immunohistochemical analyses of TLE1 and β-catenin and conducted a mutational analysis of the
CTNNB1 gene in BCA and BCAC cases to identify an approach to differentiate BCA from BCAC.
Discussion
Wnt/β-catenin signaling regulates postnatal development and regeneration of salivary gland and it is under in tight control because the dysregulation of it correlates with tumorigenesis [
16,
17]. TLE1 functions in the “off” state in the Wnt/β-catenin signaling pathway [
15]. In normal salivary glands, TLE1 showed staining for acinar and intercalated ductal cells, indicating that TLE1 expression was limited to “luminal” cells. β-catenin is generally expressed in the cytoplasm or cytomembrane in normal salivary grands [
2], in contrast to the nuclear expression for TLE1, suggesting that salivary glands are normally in “off” state of Wnt/β-catenin signaling. BCA showed positive TLE1 expression in the luminal cells of glandular structures, regardless of the tumor growth patterns. This result is consistent with the theory that BCA originates from intercalated duct lesions [
7], and this finding might suggest that tumor cells maintain an “off” state of the Wnt/β-catenin signaling pathway. The staining intensity of luminal cell staining of TLE1 in normal salivary gland and BCA cases was reduced with an increase in the age of the FFPE sample, possibly indicating that the staining was naïve expression. Interestingly, the TLE1 and β-catenin staining patterns were clearly separated (TLE1 for luminal cells and β-catenin for abluminal cells). Kawahara et al. comprehensively analyzed the β-catenin expression profile in salivary gland neoplasms, which showed a nuclear expression of basaloid myoepithelial cells that was exclusively found in BCA cases, and 52% of BCA cases had activating mutations of the
CTNNB1 gene [
2]. Our results were consistent with the findings of previous reports regarding the nuclear expression of β-catenin and mutation of the
CTNNB1 gene identified in 74 and 34% of BCA cases, respectively; however, our mutation detection rate was slightly lower than their findings [
2,
3,
8].
By contrast, the BCAC cases showed TLE1 staining for luminal cells, similar to that in BCA, in non-infiltrative areas but indistinct staining in infiltrative areas. BCAC is considered to arise mostly de novo or to progress from BCA in some cases [
4,
5,
10]. TLE1 staining revealed antecedent BCA areas in all cases of BCAC. In this study, we could identify the BCAC, which some authors recognized as carcinoma ex monomorphic adenoma, carcinoma in basal cell adenoma, and BCAC evolution from a preexisting BCA [
5,
10]. In BCAC, the obscure expression of TLE1 in infiltrative areas might suggest that these areas shifted the Wnt/β-catenin signaling pathway to the “on” state compared with non-infiltrative areas, resulting in malignancy characteristics such as invasion. Indistinct staining of αSMA lining myoepithelial cells in the invasive lesions supported this suggestion because the surrounding myoepithelial cells were considered tumor suppressors and were seen in both benign and in-situ salivary gland neoplasms but were rarely found in invasive neoplasms [
18]. In BCAC, the nuclear expression of β-catenin in abluminal cells could be seen in both areas, and 50.0% (2/4) of the cases had
CTNNB1 gene mutations. The LCM allowed the separate collection of infiltrative and non-infiltrative areas, and this is the first report to document the same mutation of the
CTNNB1 gene in both areas. This finding strengthened the secondary theory and indicated that the adenoma-carcinoma sequence existed between BCA and BCAC.
Several features and immunohistochemical markers of preferentially suggestive BCAC have been proposed, although many of them remain ancillary observations [
5‐
7]. BCAC displays various growth patterns (tubular, trabecular, solid), similar to BCA; however, many authors noted that solid patterns were frequently seen in BCAC cases [
5‐
7,
10]. Nagao et al. found that BCAC cases showed increased mitotic figures (> 4/10 HPF) and a Ki67 LI (> 5%), and Wilson et al. suggested that mitotic rates above 3 per 10 HPF were more correlated with BCACs cases [
5,
7]. Most of our BCAC cases showed solid growth patterns (3/4, 75%) and satisfied the condition of an increased mitotic count (> 3/10 HPF) and a Ki67 LI (> 5%). These findings are optional but helpful for identifying BCAC. Staining for TLE1 can be an additional approach for differentiating BCAC from BCA, especially indistinct luminal cell expression for TLE1 in invasive areas of BCAC.
Our study has limitations. The number of BCAC cases was small. Further studies are warranted to confirm the utility of TLE1 immunohistochemistry in salivary gland tumors.
Conclusion
In conclusion, we successfully investigated the TLE1 and β-catenin expression profiles and the CTNNB1 gene mutational status among BCA and BCAC cases. In BCA cases, TLE1 showed staining in luminal cells in contrast to β-catenin staining in abluminal cells. In BCAC, TLE1 showed luminal cell staining in non-infiltrative areas and indistinct staining in infiltrative areas. CTNNB1 mutations were found in 34% of the BCA cases, 50% of the BCAC cases. We furthermore demonstrated the same mutation found in non-invasive and invasive areas in BCAC and indicated that the adenoma-carcinoma sequence exsisted between BCA and BCAC. Immunohistochemical analysis for TLE1 can help identify BCA and BCAC from its luminal cell staining difference, especially indistinct luminal cell expression for TLE1 in invasive areas of BCAC. Moreover, TLE1 can be luminal/ductal cell markers.