Cytopathology of benign sebaceous salivary gland neoplasia: Comparison of two analogous yet dissimilar entities

Benign sebaceous salivary gland (SG) neoplasms represent approximately 0.2% of all salivary gland neoplasms. Not only are fine needle aspiration (FNA) biopsy findings of sebaceous adenoma (SA) and sebaceous lymphadenoma (SLA) limited, but their findings are also rarely compared with one another.

lesions. 3 SA and SLA are both rare benign salivary gland (SG) neoplasms that most commonly arise in the major glands, with the parotid being the primary site for 53% of cases of SA and 73% of SLA.
Epidemiologically, the two tumours are strikingly similar, with an identical incidence of 0.1% for all SG neoplasms and slightly less than 0.5% for all SG adenomas.Additionally, there is a nearly identical male-to-female ratio of 4:3 and a mean age in the 6th decade at the time of clinical presentation. 1,2stologically, both tumours are circumscribed, often encapsulated, and composed of glandular and ductal elements showing sebaceous differentiation.Both may display varying degrees of cystic change, and both lack cytological pleomorphism.However, SA consists of solid and cystic lobular epithelial cell nests surrounded by a fibrous stroma with minimal, if any, inflammatory infiltrate.
Epithelial cells show variable degrees of sebaceous differentiation, most notably centrally located in these solid islands, and may also show foci with squamous differentiation.In contrast, SLA contains a markedly hyperplastic lymphoid stroma, often with secondary lymphoid follicle formation and germinal centres that surround and sometimes overshadow the islands of epithelial cells. 3Epithelial cell nests in SLA may be smaller than those encountered in SA and exhibit greater squamous differentiation, with sebaceous differentiation more inconspicuous compared to SA.
Due to their rarity, preoperative fine needle aspiration (FNA) biopsy of either neoplasm is infrequently encountered.No case series of SA cytopathology could be found in our review of the Englishlanguage literature.Nonetheless, a single multi-institutional case series (nine cases) 4 and a number of single and dual case reports of SLA have been reported.  We esent the FNA biopsy findings from two examples of benign sebaceous neoplasia-one SA and one SLA-and discuss the cytomorphological features and diagnostic challenges associated with each.

| MATERIAL S AND ME THODS
A review was made of our cytology and surgical pathology databases for cases interpreted as SA, SLA, and sebaceous lesion.Cut section of resected parotid gland shows a 1.9 cm circumscribed tan-yellow, soft, friable nodule without necrosis or haemorrhage within the left parotid gland (Figure 1).An FNA performed at an outside hospital was interpreted as foamy macrophages and benign epithelial cells.After transfer to our medical centre, smears from this FNA were reinterpreted as a sebaceous neoplasm.Subsequent total parotidectomy confirmed the diagnosis of sebaceous adenoma.

| Case 2
A previously well 67-year-old woman presented with a 2.3 cm right parotid mass discovered on CT imaging which demonstrated a homogenously enhancing 1.9 cm mass of the superficial lobe (Figure 2).
Her past medical history revealed diagnoses of multiple thyroid nodules, rheumatoid arthritis, osteoarthritis, obesity, and hepatic steatosis.FNA of the parotid mass was interpreted as a basaloid neoplasm with a differential diagnosis including basal cell adenoma, basal cell adenocarcinoma, cellular pleomorphic adenoma, and solid variant of adenoid cystic carcinoma.A subsequent superficial parotidectomy showed a 4.8 cm sebaceous lymphadenoma.

| Cytopathological and immunohistochemical findings
FNA smears from the sebaceous adenoma (case #1) were moderately to highly cellular and were populated by monotonous polygonal cells in clusters and as single forms.At low magnification a relatively uniform sebaceous cell (sebocyte) population was seen.These polygonal cells had the superficial appearance of foamy macrophages due to a moderate to sometimes abundant conspicuously micro-/ macro-vacuolated cell cytoplasm.Binucleated and much larger multinucleated forms were also common, with the latter containing as many as 15-20 uniform smoothly contoured nuclei.Mononuclear cells had rounded nuclei, indistinct nucleoli, and evenly dispersed nuclear chromatin.Most cells contained variably clear back-to-back cytoplasmic vacuoles, but cell cytoplasm varied from being finely granular to occasionally harbouring large vacuoles.Mucin was not seen in any of these vacuoles.Cell borders were partially to sometimes well defined.Notably, a second cell population was absent.
The smear background was clean except for red cells, and both mitotic figures and necrosis were completely absent.Tissue sections from the subsequent superficial parotidectomy showed well-defined epithelial cell nests with florid sebocytic differentiation (Figure 3).Slides from the sebaceous lymphadenoma (case #2) were completely different from those of SA, being populated primarily by small lymphocytes and occasional dendritic-lymphocytic aggregates.Only a minor population of widely scattered small clusters (< 50-100 cells per cluster) of basaloid epithelial cells was seen in these smears.
These possessed uniform rounded to oval smoothly contoured nuclei with dense hyperchromatic nucleoplasm, indistinct nucleoli, and a high nuclear/cytoplasmic ratio.Cells exhibiting sebaceous differentiation were rare and were grouped either in very small aggregates (< 10 cells), or as single cells.Similar to SA, tissue sections of SLA also showed well-defined epithelial cell islands, but these were set in a dense lymphocytic background; sebaceous differentiation was not as abundant as seen in sections of SA (Figure 4).

| DISCUSS ION
Although sebaceous glands have been described in normal SG tissue, 30 and as metaplastic foci in some SG neoplasms, [31][32][33][34][35] and even cytologically in SG tumours with adnexal differentiation, 36,37 only two benign sebaceous SG neoplasms-SA and SLA-are recognised by the WHO.We present in this report an example of the cytopathology from both types of sebaceous SG neoplasm.The SA case was misdiagnosed as a benign non-neoplastic lesion at an outside hospital.Presumably, sebocytes were mistaken for foamy macrophages.Upon receiving the cell block in this case, which had only minuscule clusters of cells, we nonetheless performed IHC staining, which proved the epithelial, non-histiocytic nature of these vacuolated cells (cytokeratin AE1/AE3, EMA positive; CD68 negative).Thus, we were able to recognise the aspirate as a sebaceous tumour.
Though no cytological atypia was seen, we used the generic term "neoplasm" rather than "adenoma" in the diagnostic line since a tumour capsule could not be evaluated using FNA, and we could not There is a marked paucity of prior cytological reports regarding SA.We could find only three prior reports where the authors provided a cytological diagnosis and demographic information, and in only two of these cases was the FNA cytopathology described.One was specifically diagnosed as SA, one as mucinous cystic lesion, and one as retention cyst (Table 1).The case of adenoma of parotid gland with sebaceous and oncocytic features by Cameron et al. merely mentions the FNA diagnosis and illustrates the histopathology, but does not discuss or illustrate the cytopathology.In addition to mistaking sebocytes for foamy macrophages or benign cyst contents, the major primary SG neoplasm in the differential diagnosis would seem to be low-grade mucoepidermoid carcinoma (MEC).This was certainly the primary lesion considered by Apple et al and explains their diagnosis of mucinous cystic lesion. 5Interestingly, Hayes et al reported an example of MEC with sebaceous differentiation. 38Unlike SA, most aspirates of low-grade MEC typically possess a background composed of thick streaks of mucin, and the vacuolated mucocytes (goblet cells) characteristically appear as univacuolated mucin-filled cytoplasm, sometimes with a signet-ring shape, rather than as cells with semi-transparent "bubbly" cytoplasm as seen in SA. 3 In troublesome cases, a cell block or smear stained with a mucicarmine stain would resolve the dilemma.Adipophilin or perilipin IHC staining is also helpful in recognising sebaceous differentiation.Secretory carcinoma (SC) is another vacuole-rich primary SG neoplasm.Unlike SA, however, FNA smears of SC typically contain a variable amount of proteinaceous background stroma, show a papillary or micropapillary architecture in smears, and exhibit cytoplasmic vacuoles that typically lack the "foamy" multivacuolated nature seen in SA. 39,40 Positive IHC staining with S-100, mammaglobin, and MUC4, 41 and if needed, analysis for ETV6 gene rearrangement allows for a specific interpretation of SC.Distinguishing SA from primary SG sebaceous carcinoma (SCA) rests on the complete lack of cytomorphological atypia in the former, whereas in poorly-differentiated forms of SCA, large misshapen nuclei, coarse nucleoplasm, and macronucleoli are present.3][44] For this reason, we believe that making a cytological diagnosis of sebaceous neoplasm is more prudent than issuing a specific diagnosis of SA in the primary setting.
In the case of SLA, a diagnosis of basaloid neoplasm was made due to the paucity of epithelial cells, and a differential diagnosis was provided, which included basal cell carcinoma, basal cell adenocarcinoma, and solid variant of adenoid cystic carcinoma.These entities include basal cell adenoma/adenocarcinoma, cellular pleomorphic adenoma, solid variant of adenoid cystic carcinoma, epithelial-myoepithelial carcinoma, and polymorphous adenocarcinoma, as well as deeply seated cutaneous tumours such as Merkel cell carcinoma, basal cell carcinoma, and pilomatrixoma that may mimic a primary SG tumour.
Compared to all SG neoplasms, those with basaloid features are more apt to be classified into either the salivary gland neoplasm of uncertain malignant potential (SUMP) category or the suspicious for malignancy category using the Milan system for reporting SG cytopathology (MSRSGC). 47Interestingly, this system fails to even list SLA in either of these categories, 48   with demographic data shows that only 3 were specifically interpreted as SLA, 7,19,26 and 2 as possible SLA; a summary is shown in Table 2. 11,13 Another SLA series of two cases from Vazmitsel et al is not included in this table as they did not specifically declare their FNA interpretation for each case, nor did they include patient demographic information. 14Applying MSRSGC to Table 2 (including our case) shows that SLAs were most likely to be classified as a benign neoplasm (35%), followed by non-neoplastic (32%); this is understandable due to the hypercellular and often overwhelming population of lymphocytes.SUMP (21%), and non-diagnostic (6%) were the next most frequent MSRSGC categories.For benign neoplasms there was an almost equal distribution among pleomorphic adenoma, Warthin tumour, and SLA/favour SLA.Only two SLA cases were diagnosed as malignant cytologically.However, the case of Mayorga et al is questionable, as both an SLA and acinic cell carcinoma were adjacent to one another upon resection.It therefore remains unclear from their report which neoplasm was actually aspirated and interpreted, but it is likely that it was the carcinoma (Table 2). 18 summary, we present single FNA biopsy examples of SA and SLA, documenting their superficial cytomorphological similarity secondary to sebocytic differentiation.Cumulatively, however, their FNA cytomorphology is vastly different due to the overwhelming lymphoid population in SLA.Because SA cannot be distinguished reliably from low-grade SCA, and because FNA may miss or incompletely sample the epithelial component of an SLA, we believe that if either of these entities is suspected cytologically, they should be placed under the SUMP category in the Milan system. 48

1 A
Non-SG lesions were excluded from this study.Patient demographics, clinical presentation, and cytopathological diagnoses were recorded.A requirement for study inclusion was that all cytology cases had a confirmatory histopathological diagnosis of SA or SLA.Percutaneous palpation-guided FNA biopsy was performed on parotid nodules using standard technique with 21-gauge needles without image guidance.Three to four passes were made into the mass, and each needle pass was rinsed into RPMI-1640 balanced salt solution after expelling the material onto glass slides to create conventional smears.No liquid-based slides were made.All smears were air-dried.Immediate microscopic assessment was made on half the smears after staining with a Romanowsky-based stain.Subsequent Papanicolaou staining was performed on the remaining half after rehydration and alcohol fixation.Formalin-fixed paraffinembedded cell blocks were prepared in all cases using the plasma thrombin technique from cells captured in the RPMI needle-rinsed solution.These were stained with haematoxylin and eosin.A panel of immunohistochemical (IHC) markers was performed if cellular material was present in the cell block.IHC staining was performed using standard heat-induced epitope retrieval methodology and commercially available antibodies.No corresponding core needle or incisional biopsies were obtained at the time of FNA for any case.Publicly available electronic databases (ie PubMed and Medline) were searched for previously reported examples of SA and SLA with FNA cytopathological findings.Only articles written in English, including single case reports and case series, with a histopathological diagnosis of SA and SLA of SG origin were considered for inclusion.78-year-old woman presented with a 1-year history of a left-sided parotid mass.Her past medical history revealed prior diagnoses of hemochromatosis, atrial fibrillation, pacemaker placement, dermatomyositis, and a distant history of cutaneous squamous cell carcinoma of the back.Physical examination showed a firm 2.4 cm left parotid mass.Positron emission tomography/computed tomography (PET/CT) revealed a posterior parotid mass with a standardised uptake value (SUV) of 39.4, and ultrasound showed a hypoechoic mass F I G U R E 1 Sebaceous adenoma.(A) Positron emission tomography/computed tomography scan demonstrates a welldemarcated posterior parotid mass; standardised uptake value = 39.4.(B) Only a small number of cells were present for ancillary testing in the cell block in case #1.These showed positive IHC staining with cytokeratin AE1/AE3 and EMA.Staining was negative for CEA, androgen receptor, CAM 5.2, podoplanin, and CD68.However, IHC applied to the resected tissue showed positive but focal staining with androgen receptor, as well as diffuse expression with multiple keratin stains.No ancillary IHC staining was performed for case #2.

F I G U R E 2
Sebaceous lymphadenoma.Computed tomography scan shows a homogenously enhancing mass at the superficial lobe of the right parotid gland discount the possibility of a capsular-penetrating low-grade sebaceous adenocarcinoma.
IHC staining was therefore not attempted.Without any evidence of cytodifferentiation, and in the absence of any background extracellular material, primary SG neoplasms that fall under the category of "basaloid neoplasm" are among the most difficult, if not the most F I G U R E 3 Sebaceous adenoma.(A) A uniform population of sebocytes is distributed in variably-sized clusters and as single cells.Note the clean but bloody background.Papanicolaou stain.(B) Sebocytes can strongly mimic foamy histiocytes, as initially occurred in this case.Note the 'frothy' background associated with the almost universally vacuolated cells.Romanowsky stain.(C) High magnification shows two large multinucleated sebaceous cells containing innumerable semi-transparent cytoplasmic vacuoles of varying sizes.Smaller mononuclear cells have finely granular cytoplasm.Rounded to oval cell nuclei display small nucleoli but no atypical features.Papanicolaou stain.(D) Resection specimen shows well-delimited nests of duct cells with obvious sebaceous differentiation.Multinucleated cells imitate those identified on smears.Haematoxylin and eosin stain difficult, to segregate from one another.
yet cells with F I G U R E 4 Sebaceous lymphadenoma.(A) Low magnification shows a single tight cluster of basaloid cells surrounded by numerous small lymphocytes.Papanicolaou stain.(B) Basaloid cells have a modest amount of non-vacuolated cytoplasm.Lymphocytes are principally small and uniform.Romanowsky stain.(C) Two rare clusters of cells exhibit sebaceous differentiation with delicate 'foamy' cytoplasmic vacuolisation.Papanicolaou stain.(D) Hyperchromatic basaloid cells display high nuclear/ cytoplasmic ratios and finely granular nucleoplasm.Romanowsky stain.Inset: Islands of basaloid cells are surrounded by a dense lymphocytic population.Focal sebaceous differentiation exists in some, but not all cell nests.Haematoxylin and eosin stain TA B L E 1 Clinical data and cytological diagnoses of SG sebaceous adenoma: current and prior studies (N = 4).