Abstract
Purpose
BSND is a chloride channel subunit that is expressed in the normal salivary gland. We aimed to validate the utility of BSND immunohistochemistry in the differential diagnosis of oncocytic salivary gland neoplasms.
Methods
BSND immunohistochemistry was performed in a retrospective cohort of 93 salivary gland lesions, enriched with tumors with oncocytic features and histologic variants of mucoepidermoid carcinoma (MEC).
Results
All oncocytomas (n = 18) showed diffuse membranous BSND immunopositivity. Warthin tumors (n = 18) were also positive for BSND, but the staining pattern was patchy cytoplasmic and membranous in 10–25% of tumor cells. Using a threshold of 10% BSND-positive cells, all Warthin tumors were positive, while none of Warthin-like MECs or non-MEC salivary tumors were positive. Applying the same 10% positivity criterion, two oncocytic MECs were positive for BSND. The percentage of BSND staining in oncocytic MECs was up to 20%. In contrast, BSND was diffusely positive in oncocytomas with a median percentage of positivity of 95% (range: 40 − 100%). Therefore, a higher threshold of > 20% BSND-positive cells may be considered when differentiating between oncocytoma and oncocytic MEC.
Conclusion
BSND immunohistochemistry is a potentially useful diagnostic marker for salivary gland neoplasms, especially oncocytic and Warthin-like MECs. A threshold of ≥ 10% positivity can differentiate Warthin tumors from Warthin-like MECs, whereas > 20% positivity can be effective for separating oncocytomas from oncocytic MECs.
Keywords: BSND Immunohistochemistry, Oncocytoma, Warthin Tumor, Mucoepidermoid Carcinoma
Introduction
Barttin CLCNK type accessory subunit beta (BSND) is a gene located on chromosome 1p, encoding an essential beta subunit of CLC chloride channels [1, 2]. Germline alteration of the BSND gene can lead to infantile Bartter syndrome with sensorineural deafness (SND), an autosomal recessive salt-losing neuropathy [1, 2].
Recently, BSND immunopositivity has been described in the loop of Henle of the kidney and striated duct of the salivary gland [3, 4]. Furthermore, BSND immunoexpression has been proposed as a promising diagnostic marker for renal oncocytoma and chromophobe renal cell carcinoma [3]. In 2018, Shinmura et al. reported BSND immunopositivity in 25 Warthin tumors and four oncocytomas [4]. Other tested salivary gland neoplasms, including adenoid cystic carcinoma, acinic cell carcinoma, mucoepidermoid carcinoma, and salivary duct carcinoma, were negative for BSND. Based on their findings, the authors proposed BSND immunohistochemistry as a novel diagnostic marker for oncocytic salivary gland neoplasms. However, no further studies have been published since to validate the utility of BSND immunohistochemistry in diagnosing salivary gland neoplasms.
Beyond oncocytoma and Warthin tumor, many salivary gland neoplasms, such as pleomorphic adenoma, epithelial-myoepithelial carcinoma, myoepithelial carcinoma, acinic cell carcinoma, mucoepidermoid carcinoma (MEC), striated duct adenoma, and polymorphous adenocarcinoma, may exhibit focal or diffuse oncocytic changes [5–11]. Furthermore, several additional tumor types characterized by eosinophilic tumor cells may be considered in the differential diagnoses of oncocytic salivary gland neoplasms, such as secretory carcinoma, apocrine intraductal carcinoma, and salivary duct carcinoma [9]. Given that a wide spectrum of salivary gland neoplasms, whether benign, low grade malignant, or high grade malignant that may exhibit oncocytic/oncocytoid features, there is a need for novel diagnostic markers to accurately differentiate these tumors, especially in small biopsy material. This need seems to be particularly critical in the case of differentiating oncocytic and Warthin-like mucoepidermoid carcinoma from benign oncocytoma and Warthin tumor.
In this study, we aimed to validate BSND as a potential diagnostic immunomarker in 92 cases of salivary gland lesions, focusing particularly on its utility in differentiating oncocytic or Warthin-like MECs from their diagnostic mimickers.
Materials and methods
Study Cohort
BSND immunohistochemistry was performed on 92 cases of salivary gland lesions, including 20 Warthin tumors, 18 oncocytomas, 23 MECs, 13 pleomorphic adenomas, four acinic cell carcinomas, three epithelial-myoepithelial carcinomas, three polymorphous adenocarcinomas, three salivary duct carcinomas, one basal cell adenoma, one secretory carcinoma, and one intraparotid dermoid cyst. Among the 23 MECs, eight were Warthin-like subtype and six were oncocytic subtype. Oncocytic features were noted in four out 13 pleomorphic adenomas.
BSND Immunohistochemistry and Interpretation
BSND immunohistochemistry was performed using a BSND monoclonal antibody (Clone A-3, dilution 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) on the Leica Bond III automatic platform (Leica Biosystems, Wetzlar, Germany). The percentage of positive tumor cells, the staining intensity, and the staining pattern (being cytoplasmic, membranous, or both) were evaluated by a head and neck pathologist (NK). A representative tumor section was stained per tumor. The percentage and intensity of BSND positivity were evaluated on all tumor cells throughout the entire section. BSND positivity was defined using two cut-off values: 1) any cytoplasmic and/or membranous positivity in tumor cells, and 2) ≥ 10% positive tumor cells as defined previously by Shimura et al. [4]. The percentage of positivity was calculated for each tumor.
Results
BSND selectively labels striated and interlobular ducts of salivary glands
In normal salivary glands, BSND immunopositivity was only seen in the striated ducts (Fig. 1A and B) and interlobular ducts. Other structures, e.g. acini and intercalated ducts, were negative for BSND. The staining pattern in the striated and interlobular ducts was diffuse and membranous. When positive, BSND showed strong staining intensity in the non-neoplastic and neoplastic salivary gland lesions.
Fig. 1.
BSND immunohistochemistry in the salivary gland and salivary gland neoplasms. (A-B) BSND selectively labels striated ducts (S) and interlobar ducts (top left) of the salivary gland, while acini (A) and intercalated ducts (I) are negative for BSND. (C-D) An oncocytoma exhibits diffuse and strong membranous BSND immunopositivity. (E-F) An oncocytic MEC with MAML2 fusion shows very focal BSND immunopositivity in 3% of tumor cells. (G-H) Scattered patchy (20%) BSND positivity observed in a Warthin tumor, which lacks MAML2 translocation. (I-J) A Warthin-like MEC with MAML2 fusion shows cytoplasmic and membranous BSND immunopositivity in rare (< 1%) tumor cells. (K-L) A pleomorphic adenoma with oncocytic changes is entirely negative for BSND with the myxoid stroma visible within the tumor (top right)
BSND Immunostaining in MEC
The study included 23 MECs. Among these, six were classified as oncocytic subtype, defined as tumors with ≥ 50% oncocytic cells, and eight were classified as Warthin-like subtype, defined as MECs with a prominent lymphoid stroma, predominantly cystic, and exhibiting multifocal oncocytic changes, mimicking Warthin tumor.
Eight cases were examined for MAML2 fusion using either fluorescence in situ hybridization or ARCHER RNA sequencing as described previously [12]. Among these, seven harbored MAML2 fusion, including four Warthin-like MECs, one oncocytic subtype, and two classic MECs. One classic MEC did not show MAML2 fusion.
When defining BSND immunopositivity as any cytoplasmic or membranous staining, nine MECs (39%, Table 1) were positive for BSND. Among these, half of the Warthin-like and oncocytic subtypes were positive (Warthin-like: 4/8; Oncocytic: 3/6), whereas 22% (2/9) of the classic MECs were positive. All positive cases showed only focal cytoplasmic and membranous staining in 0.5–20% of tumor cells. The median percentage of BSND-stained tumor cells was 3%.
Table 1.
BSND immunohistochemistry in salivary gland neoplasms. cyto.: cytoplasmic, mem.: membranous, both: both cytoplasmic and membranous, EMC: epithelial-myoepithelial carcinoma, PAC: polymorphous adenocarcinoma, NA: not applicable
| Diagnosis | BSND positivity (any stain) | BSND positivity (≥ 10% tumor cells) | % of positive tumor cells Median (range) | Staining pattern | ||
|---|---|---|---|---|---|---|
| Cyto. | Mem. | Both | ||||
| Warthin tumor | 20/20 (100%) | 20/20 (100%) | 20 (10–25) | 0 (0%) | 0 (0%) | 20 (100%) |
| Oncocytoma | 18/18 (100%) | 18/18 (100%) | 95 (40–100) | 0 (0%) | 18 (100%) | 0 (0%) |
| Mucoepidermoid carcinoma | 9/23 (39%) | 2/23 (9%) | 3 (0.5–20) | 0 (0%) | 0 (0%) | 9 (100%) |
| Classic | 2/9 (22%) | 0/9 (0%) | 3 (1–5) | 0 (0%) | 0 (0%) | 2 (100%) |
| Warthin-like | 4/8 (50%) | 0/8 (0%) | 1.5 (0.5-3) | 0 (0%) | 0 (0%) | 4 (100%) |
| Oncocytic | 3/6 (50%) | 2/6 (33%) | 15 (3–20) | 0 (0%) | 0 (0%) | 3 (100%) |
| Pleomorphic adenoma | 4/13 (31%) | 0/13 (0%) | 3 (0.5-5) | 2 (50%) | 0 (0%) | 2 (50%) |
| With oncocytic features | 1/4 (25%) | 0/4 (0%) | 5 | 0 (0%) | 0 (0%) | 1 (100%) |
| Acinic cell carcinoma | 0/4 (0%) | 0/4 (0%) | NA | NA | NA | NA |
| EMC | 1/3 (33%) | 0/3 (0%) | 5 | 0 (0%) | 0 (0%) | 1 (100%) |
| PAC | 0/3 (0%) | 0/3 (0%) | NA | NA | NA | NA |
| Salivary duct carcinoma | 0/3 (0%) | 0/3 (0%) | NA | NA | NA | NA |
| Adenoid cystic carcinoma | 0/2 (0%) | 0/2 (0%) | NA | NA | NA | NA |
| Basal cell adenoma | 0/1 (0%) | 0/1 (0%) | NA | NA | NA | NA |
| Dermoid cyst (intraparotid) | 0/1 (0%) | 0/1 (0%) | NA | NA | NA | NA |
| Secretory carcinoma | 0/1 (0%) | 0/1 (0%) | NA | NA | NA | NA |
However, when using a threshold of ≥ 10% BSND positive tumor cells as positive, only two MECs were positive for BSND, both of which were of oncocytic subtype.
BSND is Differentially Expressed in Oncocytoma and Oncocytic MEC
All oncocytomas (n = 18) were BSND-positive using either any positivity or the 10% cut-off value, showing a membranous staining pattern. One oncocytoma case with clear cell changes showed BSND-positivity in 40% of tumor cells, while the remaining 17 cases had diffuse BSND membranous staining in at least 85% of tumor cells (Fig. 1C and D). The median percentage of tumor cells positive for BSND was 95%.
In contrast, BSND immunostain was either entirely negative (n = 3) or focal (n = 3) in MEC. The median percentage of BSND-stained tumor cells was 15%, ranging from 3 to 20% (Fig. 1E and F). BSND immunopositivity in oncocytic MEC was in noted in the oncocytic area in a haphazard (random) pattern. Taken together, a threshold of > 20% BSND positivity is required to definitively differentiate between oncocytoma and oncocytic MEC.
BSND Immunoexpression in Warthin Tumors and Warthin-like MECs
All Warthin tumors (n = 20) were positive for BSND using either positivity criterion (any positivity or the 10% cut-off). The staining pattern was cytoplasmic and membranous in scattered tumor cells, ranging from 10 to 25% (Median 20%). Among these, one Warthin tumor with 20% BSND immunopositivity was examined using FISH technique and found negative for MAML2 translocation (Fig. 1G and H).
In comparison, Warthin-like MECs were either entirely negative (n = 4) or showed very focal BSND staining in up to 3% of tumor cells (n = 4). The median percentage of BSND positive tumor cells in these cases was 1.5% (range: 0.5–3%, Fig. 1I and J).
Using the 10% cut-off for BSND positivity, all Warthin tumors were positive for BSND, while all Warthin-like MECs were negative.
BSND in Other Salivary Gland Lesions
Focal BSND positivity in 0.5–5% of tumor cells was seen in 31% (4/13) of pleomorphic adenoma, including 25% (1/4) of those with oncocytic features (Fig. 1K and L), and 33% (1/3) of epithelial-myoepithelial carcinoma. The staining pattern appeared to be random (haphazard). The other tested lesions, including acinic cell carcinoma (n = 4), polymorphous adenocarcinoma (n = 3), salivary duct carcinoma (n = 3), basal cell adenoma (n = 1), intraparotid dermoid cyst (n = 1), and secretory carcinoma (n = 1), were entirely negative for BSND.
When using a ≥ 10% positive tumor cells as cut-off value for BSND positivity, none of these tested tumors were considered positive for BSND.
Sensitivity and Specificity of BSND Immunohistochemistry
When applying a threshold of ≥ 10% positive tumor cells for BSND immunopositivity, the sensitivity and specificity of BSND in diagnosing salivary gland oncocytomas and Warthin tumors were 100% and 96% respectively. The only false-positive cases were two oncocytic MECs.
Discussion
The differential diagnoses of oncocytic/oncocytoid salivary gland neoplasms are broad, involving benign, low grade malignant, and high-grade malignant tumors. These tumors include oncocytoma, Warthin tumor, pleomorphic adenoma with oncocytic changes, striated duct adenoma, oncocytic myoepithelial carcinoma, oncocytic and Warthin-like MEC, oncocytic epithelial-myoepithelial carcinoma, apocrine intraductal carcinoma, secretory carcinoma, and salivary duct carcinoma [5–11]. Definitive distinction among these tumors, especially in small biopsy, can be challenging, and may require extensive work up, including immunohistochemistry and molecular testing for diagnostic fusions or mutations [9]. Typically, MEC is positive for CK5/6, p63, and p40, and shows mucin staining in the mucinous component of the tumor. However, these immunohistochemical markers lack specificity and may not consistently exhibit diffuse staining. Additionally, mucocytes can be seen in other types of salivary gland tumors including Warthin tumor. Oncocytic and Warthin-like MECs, in particular, may closely mimic oncocytomas and Warthin tumors histologically, and may only be distinguished by the presence of focal classic MEC areas and/or the detection of MAML2 fusion. Accurate differentiation is crucial as oncocytoma and Warthin tumor are benign, exhibiting different clinical behavior and requiring different management approaches.
In 2018, Shinmura et al. proposed BSND immunohistochemistry as a novel diagnostic immunomarker for oncocytic salivary gland neoplasms [4]. In their study, BSND immunopositivity, defined as ≥ 10% of tumor cells with BSND cytoplasmic or membranous immunostain, was only seen in Warthin tumor (n = 25) and oncocytoma (n = 4). None of the other five types of salivary gland tumors tested, including pleomorphic adenoma (n = 11), adenoid cystic carcinoma (n = 7), acinic cell carcinoma (n = 6), mucoepidermoid carcinoma (n = 6), and salivary duct carcinoma (n = 5), showed BSND positivity. Therefore, the authors suggested BSND as an excellent immunohistochemical marker for oncocytic salivary gland tumors.
To validate their previous findings and explore the utility of BSND in distinguishing MECs, particularly Warthin-like and oncocytic subtypes from their diagnostic mimickers, we herein expanded our study to examine BSND immunoexpression in a broader spectrum of salivary gland neoplasms with oncocytic/oncocytoid features and variant histologies of MECs. The results of the current study validated the findings of Shinmura et al. [4], showing that oncocytomas and Warthin tumors were consistently positive for BSND. While oncocytomas showed diffuse membranous BSND immunopositivity, Warthin tumors exhibited scattered patterns of membranous and cytoplasmic BSND staining in 10–25% of tumor cells.
We observed that BSND immunopositivity can occur in MECs, particularly oncocytic MECs, where two of six oncocytic MECs (33%) exhibiting ≥ 10% BSND positive tumor cells; however, in these cases, BSND staining was focal with a median percentage of positive cells of 15% (up to 20%). In contrast, oncocytomas typically displayed diffuse BSND immunopositivity, with a median percentage of positive cells of 95% (range 40–100%). Therefore, diffuse BSND positivity is a characteristic feature of salivary gland oncocytomas. A cut-off value of ≥ 20% BSND-positive tumor cells may be appropriate in separating oncocytic MECs from oncocytomas.
When distinguishing Warthin-like MECs from Warthin tumors, the 10% threshold proposed by Shinmura et al. [4] appears to be effective. Using this cut-off, all Warthin tumors were positive for BSND, while all Warthin-like MECs were negative for BSND.
Lastly, it is worthwhile to note that focal, rare BSND staining (< 10% tumor cells) may occur in other salivary tumors, including four Warthin-like MECs, one oncocytic MEC, one epithelial-myoepithelial carcinoma, and four pleomorphic adenomas, including one with oncocytic features observed in the current study. This suggests that very focal (< 10% tumor cells) BSND staining can be seen in other salivary gland neoplasms, whether oncocytic or non-oncocytic. Therefore, rare focal staining (< 10%) may not be reliable for the differential diagnosis of these tumors.
In conclusion, we herein investigated BSND as a useful diagnostic immunostain for oncocytic salivary gland neoplasms, particularly in differentiating oncocytomas and Warthin tumors from oncocytic and Warthin-like MECs. BSND is diffusely positive in oncocytomas and patchy positive in Warthin tumors. A threshold of ≥ 10% BSND immunopositivity appears to be effective in separating Warthin tumors from Warthin-like MECs, whereas a higher threshold, ≥ 20%, may be necessary when differentiating oncocytomas from oncocytic MECs.
Author Contributions
Study design and conception; NKCase collection: DR, NKImmunohistochemistry: RS, AJ, DFPathology review: NKDatabase management and data analysis: BX, NKManuscript drafting: BXManuscript editing: BX, AA, DF, RS, HQ, DR, AJ, RG, NK.
Funding
Research reported in this publication was supported in part by the Cancer Center Support Grant of the National Institutes of Health/National Cancer Institute under award number P30CA008748.
Data Availability
No datasets were generated or analysed during the current study.
Declarations
Competing Interests
No competing financial interests exist for all contributory authors. The research meets the ethics guidelines, including adherence to the legal requirements of the country where the study was performed.
Footnotes
Publisher’s Note
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Data Availability Statement
No datasets were generated or analysed during the current study.