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Case Review

Ancillary Testing of Salivary Gland Fine-Needle Aspiration Biopsy Material

Wong, Kristine S. MD; Krane, Jeffrey F. MD, PhD

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doi: 10.1097/PCR.0000000000000401
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A 79-year-old man with a complicated medical history, including congestive heart failure, coronary artery disease, and chronic kidney disease, presented to the hospital with a heart failure exacerbation. In addition, he was noted to have an almost 100-lb unintentional weight loss over an 18-month period. The patient notably had a remote smoking history and prior melanoma and cutaneous squamous cell carcinoma. During his admission, a lesion on the back was noted, and biopsy revealed squamous cell carcinoma. Imaging of the neck also demonstrated a 2-cm rounded nodule arising in the superficial parotid. Given the clinical concern for malignancy, a fine-needle aspiration (FNA) was performed. Smears revealed a cellular neoplasm composed of spindled to epithelioid cells with small to intermediate amounts of cytoplasm and minimal cytologic atypia (Figs. 1A, B). In some areas, the cells were associated with metachromatic matrix material. The same population of cells was noted in the cell block (Fig. 1C), and pleomorphic adenoma gene 1 (PLAG1) immunohistochemistry (IHC) revealed strong, diffuse nuclear staining (Fig. 1D). A diagnosis of pleomorphic adenoma (PA) was made. However, per request from the clinical team, cytogenetic analysis was performed, revealing a t(3;8)(p21;q12) rearrangement, consistent with CTNNB1-PLAG1 fusion, a hallmark of PA. No additional workup or surgery was performed during the patient's extensive hospital stay, and the patient was eventually discharged home.

Diff-Quik (A) and Papanicolaou (B) stains on smear preparations of this pleomorphic adenoma demonstrate a cellular neoplasm comprised predominantly of bland spindled to plasmacytoid myoepithelial cells. In some areas, the cells were embedded within fibrillary metachromatic matrix material (A). A cell block demonstrated predominantly myoepithelial cells with scant matrix (C). Immunohistochemistry for PLAG1 was diffusely positive (D).

Salivary gland pathology can be challenging because of the relative rarity of tumors, which comprise only a small percentage of tumors of the head and neck. In a recent analysis of the incidence of major salivary gland carcinoma in the United States over a 14-year period, the incidence rate was only ~12/1,000,000 people per year.1 Additionally, the diversity of tumor types and significant morphologic overlap also contribute to challenges in diagnosis. There are currently 33 epithelial tumors (22 malignant/uncertain malignancy potential, 11 benign) recognized in the WHO Classification of Head and Neck Tumours,2 of which only a minor subset is encountered by pathologists with any frequency in day-to-day practice. However, as the understanding of the biologic and molecular pathogenesis of salivary tumors has increased over the last few decades, so too has the number of ancillary studies available. Although histochemical and immunohistochemical studies have historically had limited utility due to lack of sensitivity and specificity, molecular testing and use of IHC as a molecular surrogate have emerged as effective tools.

Most salivary tumors currently undergo FNA prior to surgical resection in the United States. When combined with imaging and clinical history, diagnostic accuracy of FNA is high, and routine use of core biopsy or frozen section is currently not recommended.3 Although most salivary tumors are resected regardless of benignancy or specific tumor type, FNA cytology is nevertheless an important tool to (1) confirm a neoplastic process, (2) identify patients who would benefit most from surgery (ie, benign vs malignant, primary vs. metastatic, epithelial vs hematolymphoid), and in some cases (3) help to determine extent of surgery. Recently, The Milan System for Reporting Salivary Gland Cytopathology (TMS) was introduced to help standardize the reporting of salivary gland FNA cytology. Within this system, there are 7 diagnostic categories (including 4 neoplastic) as follows: I—nondiagnostic, II—nonneoplastic, III—atypia of undetermined significance, IVA—neoplasm: benign, IVB—salivary gland neoplasm of uncertain malignant potential, V—suspicious for malignancy, and VI—malignant.4 Using this framework, ancillary studies may help to more accurately separate tumors into 1 of the 4 neoplastic categories, for example, downgrading or upgrading tumors from salivary gland neoplasm of uncertain malignant potential or suspicious for malignancy to benign or malignant. The TMS also recommends characterizing malignant aspirates as low grade or high grade if possible. Ancillary testing may also be of use in making this distinction in some instances.

This review provides an overview of ancillary testing that can be used in salivary gland cytology, with a focus on fluorescence in situ hybridization (FISH) and IHC. Given that tumors of the major salivary glands are much more likely to undergo FNA, tumors that occur at these sites will be emphasized. Finally, entities have been separated into 2 main diagnostic categories: (1) basaloid, which includes biphasic tumors and/or tumors that have a prominent myoepithelial component; and (2) oncocytic/clear cell, which encompasses tumors with more abundant granular, foamy, or vacuolated cytoplasm. Although salivary tumors in TMS are often discussed in the context of 3 subcategories, that is, basaloid, oncocytic/oncocytoid, and clear cell,4 tumors with oncocytic and clear cell features often overlap and have been combined here.


Pleomorphic Adenoma and Carcinoma ex Pleomorphic Adenoma

Pleomorphic adenoma is the most common salivary tumor, characterized by chondromyxoid matrix, a ductal/epithelial component, and myoepithelial cells that can have variable cytomorphology, from spindled to plasmacytoid. The relative proportion of each component as well as architectural patterns is highly variable. Although the diagnosis is often readily made on FNA when all 3 components are present, diagnosis can be challenging in matrix-poor cases. In these cases, distinction between other biphasic or basaloid/myoepithelial-predominant tumors may be difficult to impossible using only morphology. General IHC for ductal (ie, low-molecular-weight cytokeratins) or myoepithelial (ie, p40, SMA, calponin) components is also of limited benefit. However, the majority of PAs are characterized by recurrent molecular alterations, the most common of which are rearrangements involving PLAG1 on chromosome 8q12, which leads to overexpression of PLAG1.5–8 Additionally, a smaller subset of PA harbor rearrangements involving high mobility group AT-hook 2 (HMGA2) on chromosome 12q14-15.9–11

Similarly, carcinoma ex PA also demonstrates frequent PLAG1 and HMGA2 alterations.12–15 Although carcinoma ex PA often presents as a high-grade adenocarcinoma (ie, salivary duct carcinoma [SDC] ex PA, see also Salivary Duct Carcinoma), it may less commonly take the form of a myoepithelial-rich malignancy, including myoepithelial carcinoma and epithelial-myoepithelial carcinoma (EMCA).16–19 Interestingly, El Hallani et al18 found that nearly 80% of EMCAs arose from PA and that de novo EMCA (without PLAG1 or HMGA2 alterations or morphologic evidence of PA) frequently harbored HRAS mutations, consistent with prior studies demonstrating HRAS mutations in a subset of EMCA.20,21 Myoepithelial carcinoma and EMCA may be especially challenging to differentiate from benign basaloid/biphasic tumors on FNA or even surgical resection, as they are conventionally low grade and may not demonstrate foci with high-grade morphology. Xu et al,19 for example, found that a cohort of myoepithelial carcinomas (including ex PA and de novo) that had recurred or metastasized had been misdiagnosed as cellular PA or other benign neoplasm. Although ancillary studies may not help in determining malignancy, they can clarify disease pathogenesis or be used to confirm origin in the setting of metastases.

Both PLAG1 and HMGA2 rearrangements may be detected by FISH, and IHC for PLAG1 and HMGA2 has also become more widely available (Figs. 1 and 2). However, the performance of these stains is variable. In surgical pathology specimens, HMGA2 IHC demonstrates greater than 95% specificity but low sensitivity for PA,22 which is not surprising given the relatively low frequency of HMGA2 rearrangements. Conversely, PLAG1 IHC demonstrates high sensitivity (>90%) in PA,22–27 although decreased sensitivity is seen in carcinoma ex PA.12,22,26,27 Specificity of PLAG1 IHC in PA also appears to be lower than that of HMGA2, with staining reported in up to 29% of non-PA tumors.23,24,27 In the few studies that have evaluated PLAG1 IHC in cytologic material, sensitivity was 55% and 73%.28,29 However, in combination with HMGA2 IHC, sensitivity was improved to 85% in one study.28

A cell block of this pleomorphic adenoma demonstrates myoepithelial cells embedded in chondromyxoid matrix material (A). Immunohistochemistry for HMGA2 shows strong, diffuse nuclear staining (B).

Basal Cell Adenoma/Adenocarcinoma

Basal cell adenoma (BCA) and basal cell adenocarcinoma (BCAC) are morphologically identical tumors distinguished solely by the presence of invasion and are therefore indistinguishable on FNA. Although they are biphasic tumors comprised of luminal cells with slightly increased cytoplasm and abluminal basaloid/myoepithelial cells, the dual populations can sometimes be difficult to appreciate in cytologic preparations. Fine-needle aspiration of BCA/BCAC typically demonstrates a population of small- to intermediate-sized cells with hyperchromatic nuclei and scant cytoplasm, sometimes in association with dense hyaline matrix material. This hyaline matrix, however, is not specific for BCA/BCAC and may be seen in adenoid cystic carcinoma (AdCC) as well as in PA, although fibrillary matrix with cells embedded within is more common in the latter.

Most BCAs are characterized by mutations in CTNNB1, leading to nuclear overexpression of β-catenin.30,31 The membranous variant of BCA represents a distinct subtype more frequently characterized by CYLD alterations.32 The molecular alterations in BCAC are less understood and were thought to be distinct from BCA.31,33 Recently, however, CYLD mutations were detected in approximately one-third of BCAC, although mutation distribution differed from BCA, with mutations in exons 12 to 20 occurring exclusively in BCAC.34 Nuclear β-catenin expression by IHC can be variable in BCA, often with more prominent staining in the abluminal cell population.31 Although the performance of β-catenin IHC in cell blocks of BCA has not been reported to our knowledge, given this variability in staining, interpretation may be difficult in cytologic material. In addition to β-catenin, LEF-1 (lymphoid enhancer binding factor 1), a transcription factor in the Wnt signaling pathway that interacts with β-catenin, has been evaluated as a diagnostic marker. LEF-1 has been shown to be expressed in greater than 60% of BCAs, often with coexpression of β-catenin.35,36 In a study examining LEF-1 in cell block material, 60% BCA expressed LEF-1, although staining was also seen in more than half of PA. However, the vast majority (>90%) of AdCCs were negative.36

Adenoid Cystic Carcinoma

Similar to PA and BCA, AdCC is a biphasic tumor comprised of epithelial and myoepithelial cells. Tumors may have cribriform, tubular, and/or solid growth. In tumors with cribriform architecture, hyaline globules are often seen within the pseudocystic spaces and are surrounded by basaloid tumor cells. These globules are intensely metachromatic on Romanowsky stain but are more difficult to see on Papanicolaou stain. Dense hyaline matrix material surrounding cell nests may also be seen, similar to BCA, although peripheral palisading common to BCA is not a typical feature of AdCC. The basaloid cells of AdCC demonstrate hyperchromatic, angulated nuclei with more cytologic atypia compared with BCA and PA, although cells are still relatively monomorphic; pleomorphism is a feature seen more often in tumors that have undergone high-grade transformation.

Adenoid cystic carcinoma is characterized by recurrent t(6;9) rearrangements resulting in MYB-NFIB fusion in approximately 50% of cases,37–39 with a smaller percentage harboring MYBL1-NFIB [t(8;9)] fusions.40,41 Overexpression of MYB mRNA is seen in up to 97% of AdCCs including fusion-negative tumors,38,42,43 whereas protein expression by IHC is seen in up to 82% of cases39,42 (Fig. 3). Similar to the variable immunoreactivity seen with β-catenin in BCA, MYB staining is also accentuated in the abluminal myoepithelial component in AdCC,39,42 which may limit interpretation in cytologic material. Although FISH or other molecular-based testing for MYB rearrangement is specific for AdCC, MYB IHC positivity can be seen in other tumors as well. In one study, MYB IHC was positive in 14% of non-AdCC tumors including most basaloid squamous cell carcinomas evaluated as well as smaller proportions of BCA/BCAC and PA, among others.42 In another study, Pusztaszeri et al44 found MYB positivity in 20% of PA resection specimens. Additionally, few studies have examined MYB IHC in FNA material.28,44 In the aforementioned study, Pusztaszeri et al44 also performed IHC on alcohol-fixed smears and found 80% of AdCCs were positive, whereas Foo et al28 examined cell block material and found staining in only 36% of AdCCs; however, specificity was relatively high in both studies (100% and 85%, respectively). Although MYB rearrangement is detectable in only about half of cases as mentioned earlier,37–39MYB FISH may potentially still be diagnostically useful given the imperfect specificity of IHC. It has been found to have similar sensitivity and specificity in cell block material compared with surgical resection specimens and can be a valuable tool in confirming a diagnosis of AdCC on FNA.45

Basaloid cells with scant cytoplasm associated with slightly larger epithelioid cells can be seen in this cell block of AdCC (A). MYB IHC revealed strong nuclear staining (B).


Acinic Cell Carcinoma

Acinic cell carcinoma (AciCC) is a typically low-grade malignancy that consists of cells with serous acinar differentiation admixed with intercalated duct-type cells with vacuolated or clear cytoplasm. On FNA, intermediate- to large-sized cells with abundant cytoplasm can be seen, often arranged in loose aggregates or sheets. While the basophilic zymogen granules are often readily identifiable on conventional hematoxylin-eosin stain in resection specimens or cell blocks, they can be somewhat more difficult to identify in FNA, appearing blue on Papanicolaou stain and metachromatic on Romanowsky stain. Additionally, AciCCs are often associated with a prominent tumor-associated lymphoid proliferation and may have abundant lymphocytes in the background.

The zymogen granules of AciCC are PAS-positive and diastase-resistant, although tumors with less overt zymogen granules may require additional IHC workup. Although not entirely specific, DOG1 (a marker of serous acinar and intercalated duct differentiation) typically demonstrates strong apical-to-diffuse membranous staining in AciCC46–48 (Fig. 4). Similarly, SOX10 is expressed in serous acini/intercalated ducts and is frequently positive in AciCC,48–50 although SOX10 expression is also seen in secretory carcinoma (SC) and myoepithelial cell–rich neoplasms.48,50,51 In cell block material, the reported sensitivities of DOG1 and SOX10 have been modest (77% and ~62%, respectively)48 compared with greater than 90% in resections.46–49 Recently, the vast majority of AciCCs have been found to harbor recurrent t(4;9)(q13;q31) rearrangements involving nuclear receptor subfamily 4 group A member 3 (NR4A3),52 with a smaller subset having HTN3-MSANTD3 rearrangements.53–55 NR4A3 expression is seen in both fusions, with NR4A3 IHC positivity in greater than 97% of AciCCs overall.52,55 In our experience, although NR4A3 IHC is highly sensitive and specific for AciCC, rarely focal or weak staining can be seen in other salivary tumors. Performance in cell block material is also unknown, with no published studies to date. NR4A3 FISH can therefore still be a valuable tool in FNA material and/or cases with equivocal IHC.

Sheets of large cells with abundant granular, clear to slightly basophilic cytoplasm are seen in this cell block of acinic cell carcinoma (A). Immunohistochemistry for DOG1 revealed complete membranous staining around the tumor cells (B). Additionally, intracellular granules were PAS-positive/diastase-resistant, whereas S100, mammaglobin, and p63 immunostains were negative in the tumor (not shown).

Mucoepidermoid Carcinoma

Mucoepidermoid carcinoma (MEC) is the most common salivary malignancy, traditionally described as having 3 cell types: mucous, intermediate, and epidermoid, although the cytomorphology of intermediate and epidermoid cells overlaps significantly. Mucoepidermoid carcinomas have variable histologic grade, with low-grade tumors typically demonstrating minimal cytologic atypia, a greater cystic component, and higher proportion of mucous cells. Low-grade MECs are therefore a common cause of false-negative FNA diagnoses, particularly when predominantly cyst contents with minimal cellularity are aspirated.56 Mucoepidermoid carcinomas may also have a prominent tumor-associated lymphoid proliferation and can overlap with AciCC and Warthin tumor on FNA. Oncocytic, clear cell, Warthin-like, and rarely ciliated variants of MEC can also occur,57–60 contributing further to potential pitfalls in the diagnosis.

In challenging cases, mucicarmine stain can be used to confirm the presence of intracellular mucin. Additionally, MECs (including those that are oncocytic) are positive for p40/p63 (Fig. 5), in contrast to AciCC and SDC.57,61–63 However, (hyalinizing) clear cell carcinoma (CCC) is also consistently positive for p63/p40.64–67 In cases where CCC is suspected, EWSR1 FISH can confirm the diagnosis, as CCCs are characterized by EWSR1-ATF164–66 and rarely EWSR1-CREM fusions.67 Although few cases of SC have been reported as having p63 positivity,61,68 use of p40 (selective for the ΔNp63 isoform, which is more specific for the basal/myoepithelial/squamoid phenotype) can minimize nonspecific staining.68 Additionally, among benign tumors, Warthin tumor and oncocytoma can also express p63; however, staining is typically seen only in a basal distribution.62,69,70 For definitive confirmation of MEC, molecular studies may be used, as MECs are characterized by mastermind-like protein 2 (MAML2) rearrangements, with CRTC1-MAML2 fusions observed in most tumors71–73 and CRTC3-MAML2 fusions occurring in a smaller subset.74,75 Fluorescence in situ hybridization for MAML2 rearrangement can therefore be a valuable diagnostic tool in differentiating MEC and its variants from other salivary tumors.58–60,73,76

A cell block demonstrates a population of intermediate-sized cells with moderate to abundant amounts of eosinophilic cytoplasm. The cells are arranged in solid nests, with some areas suggesting cystic change (A). Immunohistochemistry for p40 was extensively positive (B), whereas S100, mammaglobin, and DOG1 were negative (not shown). A Mucicarmine stain showed extensive extracellular mucin, although only rare cells appeared to have intracellular mucin (C). A diagnosis of low-grade salivary neoplasm, favoring MEC, was made. Subsequent resection revealed an intermediate-grade MEC.

Secretory Carcinoma

Secretory carcinoma, formerly known as mammary analogue SC, is a more recently recognized entity within salivary gland, although it is morphologically and molecularly similar to its counterpart in breast. Secretory carcinoma is characterized by intermediate-sized cells with bland nuclei and foamy/vacuolated to granular eosinophilic cytoplasm. The tumor may demonstrate variable growth, including papillary, solid, and cystic/microcystic architecture, and typically has abundant extracellular eosinophilic material. On FNA, the cells are often loosely cohesive and may be arranged in aggregates or papillary structures. The extracellular secretory material is PAS positive and diastase resistant77–79; however, unlike the zymogen granules of AciCC, intracellular PAS-positive/diastase-resistant material is less commonly seen in SC and likely represents mucin globules.80–82

The immunohistochemical profile of SC is often characteristic, with positivity for S100 and mammaglobin in most cases,83–85 although 29% to 47% of cases may show only focal S100 positivity.79,85 While few tumor types such as polymorphous adenocarcinoma and less frequently AdCC may show similar coexpression of S100 and mammaglobin,86 tumor site (polymorphous adenocarcinoma occurs almost exclusively in minor salivary gland and is unlikely to undergo FNA) and morphologic features (basaloid cytomorphology of AdCC) can aid in the distinction. In addition to S100 and mammaglobin, SOX1048,50,51 and GATA387 are also positive in SC, although both can show staining in many other salivary tumors including AciCC.48,50,87 Positivity for p63 is rare,61,68 as mentioned earlier, in contrast to MEC and CCC.

Although a small subset of SC harbors ETV6-RET88 or ETV6-MET89 fusions, most demonstrate ETV6-NTRK3 fusions.77 As such, the utility of IHC for pan-Trk, which detects NTRK1, NTRK2, and NTRK3 fusions, has been assessed. Although studies in SC have been limited thus far, staining appears to be highly variable in surgical resections (sensitivity: 64%–90%, specificity: 46%–70%).90,91 The performance in cell blocks has not been reported to our knowledge. For these reasons, FISH for ETV6 rearrangement is still an important diagnostic tool, particularly in FNA material.82 Although it has been suggested that morphology and IHC (S100 and mammaglobin) alone are sufficient for diagnosis of SC on resection given the high correlation with ETV6 rearrangement,83 this has not been established yet in cytologic material, and FISH should therefore still be used to confirm the diagnosis.

Salivary Duct Carcinoma

Salivary duct carcinoma is a high-grade malignancy that may occur de novo or arise from PA.12,15 It is composed of apocrine-appearing cells with abundant granular cytoplasm and overt nuclear atypia. Although typically infiltrative, SDC often demonstrates areas that resemble apocrine ductal carcinoma in situ of the breast, including cribriform to solid nests that frequently have central necrosis. While some nests may, in fact, represent a true in situ component with a preserved layer of myoepithelial cells, many of these rounded nests are, in fact, invasive. Large pleomorphic cells with abundant granular cytoplasm admixed with necrosis, resembling a high-grade adenocarcinoma, may be seen on FNA. Although the appearance of SDC is often characteristic on resection, metastatic disease or other primary high-grade salivary tumors are more difficult to exclude by morphology in cytologic preparations.

Salivary duct carcinoma is negative for S100, SOX10, and p40/p63; although the latter can be used to highlight myoepithelial cells in an in situ component, it is negative within the neoplastic epithelial cells.92,93 Androgen receptor (AR) is the most specific IHC marker for SDCs among primary salivary tumors, as most SDCs demonstrate strong, diffuse AR positivity94–100 (Fig. 6). Although the reported positivity rates have been variable, in the largest series to date, greater than 97% of SDCs demonstrated AR expression.97 Other large, contemporary studies have also similarly demonstrated greater than 95% positivity.98,99 Molecular alterations in SDC are variable, with frequent mutations in the PI3K pathway. Approximately 30% of cases demonstrate ERBB2 amplification typically in the setting of SDC ex PA,15,101–103 with similar rates of HER2 IHC positivity.97–99,101,104 Finally, in SDC ex PA, FISH and IHC for PLAG1 or HMGA2 are positive in most cases.12–15,22,27 Although not required for the diagnosis of SDC, evidence of a precursor lesion could be helpful in reaching a definitive diagnosis, especially if clinical/radiographic information or biopsy material is limited.

Scant groups of large, pleomorphic cells with abundant granular, eosinophilic cytoplasm and prominent nucleoli are seen in this cell block of SDC (A). Androgen receptor IHC demonstrates strong nuclear staining (B).


Salivary tumors can be challenging to diagnose, particularly in FNA biopsies. Although morphologic features can overlap significantly, general categorization (ie, basaloid vs oncocytic/clear cell) in conjunction with ancillary studies can narrow the differential diagnosis. For example, a conservative panel of histochemical stains and/or general IHC in addition to more specific IHC surrogates for point mutations or chromosomal rearrangements may be used. Fluorescence in situ hybridization studies can also be performed on smear or cell block preparations to confirm the diagnosis. Although not necessary or feasible for every salivary tumor FNA, ancillary studies can refine diagnoses in the context of TMS and, more importantly, help to triage patients appropriately in their clinical management.


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ancillary testing; fine-needle aspiration; fluorescence in situ hybridization (FISH); salivary gland; immunohistochemistry; cytology

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