Diagnosis of Adenoid Cystic Carcinoma with Striking Tubular Hypereosinophilia by MYB and EWSR1 Breakapart Fluorescence In Situ Hybridization

Abstract

Background

Adenoid cystic carcinoma (AdCC), associated with MYB/MYBL1 gene rearrangements, shows epithelial and basaloid myoepithelial cells arranged in tubular, cribriform and solid patterns. Variations from this classic morphology make diagnosis challenging, necessitating molecular testing. AdCC with striking tubular hypereosinophilia (AdCC-STE) is one such recently described histological subtype.

Methods

A 52-year-old female presented with a floor of mouth swelling for two months, diagnosed elsewhere as polymorphous adenocarcinoma (PAC). A biopsy was obtained. With a diagnosis of oncocytic neoplasm, wide excision of the tumor was undertaken. Histological examination, fluorescence in situ hybridization (FISH) and ultrastructural examination were performed. Archival cases of PAC and epithelial myoepithelial carcinoma (EMC) were reviewed, and MYB immunostaining and FISH were performed to identify potential AdCC-STE cases.

Results

The excised tumor from the index patient showed bilayered tubules, micropapillae and cribriform pattern. Luminal cells with hypereosinophilic to clear cytoplasm were surrounded by flattened abluminal cells. Focally, basophilic matrix was seen within sharply demarcated pseudocystic spaces. FISH revealed MYB and EWSR1 gene rearrangements, confirmatory of AdCC-STE. Electron microscopy showed features consistent with AdCC; however, mitochondria were not prominent. Among 14 archival PACs, two showed MYB immunopositivity; one showed MYB rearrangement but was classical AdCC. Among 35 EMC, one case showed MYB immunoreactivity and eosinophilia of luminal cells but lacked MYB/MYBL1 rearrangement.

Conclusion

Awareness of unusual histological subtypes of AdCC, such as AdCC-STE, is imperative, as it may be misdiagnosed as PAC and EMC, among others. Presence of basophilic matrix and squamoid morules in a biphasic tumor even with hypereosinophilic rather than basaloid myoepithelial appearance should raise suspicion for AdCC-STE, and prompt molecular testing for confirmation. With wider accessibility, lower cost and significantly shorter turn-around-time when compared to RNA sequencing, FISH can be employed for confirmation of diagnosis, especially in low- and middle-income countries.

Introduction

Adenoid cystic carcinoma (AdCC) is one of the most common malignant neoplasms of the salivary glands, accounting for almost 25% of primary salivary gland carcinomas [1]. It occurs in both major and minor salivary glands, with the parotid being the most common location. It is the most common malignant tumor of the minor salivary glands, most frequently involving the palate, buccal mucosa, and tongue [2].

Microscopically, AdCC is an infiltrative biphasic tumor composed of ductal and myoepithelial cells arranged in cribriform, tubular, and/or solid patterns [3]. The tubular pattern is characterized by well-formed ducts and tubules lined by luminal ductal cells having eosinophilic cytoplasm and uniform round nuclei, surrounded by abluminal myoepithelial cells with scant clear cytoplasm and angulated hyperchromatic nuclei [4]. The basaloid myoepithelial cells predominate, and it may often be difficult to identify the luminal cells. The cribriform pattern displays microcytic spaces containing hyaline or basophilic myxoid matrix within tumor nests that are continuous with tumor stroma, while the solid pattern consists of sheets of basaloid tumor cells [5]. These patterns are usually present in combination and aid in histological diagnosis of AdCC. When absent or limited, it may be difficult to distinguish AdCC from other biphasic tumors, particularly on a biopsy.

Molecular characterization has identified t(6;9) and t(8;9) as the defining genetic alterations in AdCC. These rearrangements result in the fusion of MYB proto-oncogene on chromosome 6q or MYBL1 on chromosome 8q with NFIB on chromosome 9p [6–8]. These MYB/MYBL1 rearrangements can be detected by reverse-transcriptase PCR, fluorescence in situ hybridization (FISH), or next-generation sequencing (NGS)-based methods, and their identification is desirable in morphologically challenging cases.

While the classification and morphological spectrum of other salivary gland malignancies has evolved based on detection of genetic alterations, AdCC has had more or less consistent histological features, with classic basaloid and myoepithelial predominant morphology. Variations in morphology have, however, been noted recently, especially in non-major salivary gland locations. Mathew et al., in a series of three cases, described the presence of macrocystic architecture, trabecular pattern, and squamous differentiation in sinonasal and skull base AdCCs, which they referred to as metatypical AdCC, and recommended their recognition as a distinct subtype [9]. More recently, Weinreb et al. described a series of 16 cases of AdCC with striking tubular hypereosinophilia (AdCC-STE) in non-major salivary gland locations and harboring canonical as well as novel EWSR1::MYB and FUS::MYB gene fusions [10]. These cases had morphological features considerably divergent from the classical picture of AdCC that pathologists are familiar with and were retrospectively identified as AdCC following RNA-seq detection of the gene fusions. Differential diagnoses include polymorphous adenocarcinoma (PAC), mucoepidermoid carcinoma, and epithelial–myoepithelial carcinoma (EMC). We recently encountered a similar case in our diagnostic practice and confirmed it as AdCC-STE by utilizing FISH to detect MYB and EWSR1 rearrangements. As PAC and EMC are frequent in minor salivary glands/ other seromucinous sites and have overlapping histological and immunohistochemical features with AdCC, we also reviewed archival cases of PAC and EMC to identify any more cases of AdCC-STE.

Patients and Methods

Index Case

This 52-year-old female presented with complaints of a progressive swelling in the floor of mouth for two months. It was associated with occasional mild pain while eating food. There was no history of trauma or tooth extraction. She was a known hypertensive on regular treatment, and she did not consume tobacco in any form. On intra-oral examination, a 3 cm × 4 cm firm, tender, mobile mass was noted at the right side of the floor of mouth, with a 1 cm-sized ulcer over it. It extended from the right central incisor to the first molar. The mandible and tongue were not involved. A 1 × 1 cm soft, discreet, mobile lymph node was palpable in the neck at level IB. Contrast-enhanced computed tomography of the face and neck (Fig. 1) showed an ill-defined, heterogeneously enhancing mass of size 2.8 cm in the floor of mouth with inferior extension into the right sublingual space, and infiltration of the mylohyoid muscle anteriorly. It was abutting the right lateral border of tongue with loss of fat plane posteriorly. There were few suspicious lymph nodes in right level IB, the largest measuring 6.4 mm. A biopsy done at another hospital was reported as a tumor of salivary gland origin, possibly PAC. The biopsy was submitted to us for review. The patient then underwent transoral wide excision with right nasolabial flap reconstruction, along with right supra-omohyoid neck dissection. She had an unremarkable post-operative recovery.

Fig. 1.

Imaging of index case: Contrast-enhanced CT shows an ill-defined, heterogeneously enhancing mass in the floor of mouth (A), with infiltration of the mylohyoid muscle anteriorly and abutting the right lateral border of tongue (arrow, B)

Ultrastructural Examination

Ultrastructural examination was performed on formalin-fixed paraffin-embedded (FFPE) tumor tissue from the index case to determine if the hypereosinophilic cells were oncocytic in nature. FFPE tissue was deparaffinized, rehydrated, post-fixed in 1% osmium tetroxide, dehydrated, and embedded in epoxy resin. Thick sections were cut to select representative areas for ultra-thin sectioning. Ultra-thin sections were stained with uranyl acetate and lead citrate and examined under a transmission electron microscope.

Review of Archival Cases of PAC and Epithelial–Myoepithelial Carcinoma

Cases of PAC and EMC were retrieved from our departmental archives. Histological features were reviewed, and all cases were subjected to MYB immunohistochemistry (IHC) to identify the cases of AdCC-STE. MYB IHC was performed manually on whole tissue sections using a rabbit monoclonal primary antibody (clone D2R4Y, Cell Signaling Technology) in a dilution of 1:200. Nuclear staining in ≥ 10% of tumor cells was considered as positive. Ethics approval was obtained to perform these experiments on archival patient material. FISH for MYB rearrangement was performed on all EMCs in non-major salivary gland locations, and in all MYB immunopositive cases of PAC and EMC.

Fluorescence In Situ Hybridization

FISH was performed on FFPE tissue sections using commercially available probes, as described previously [11, 12]. MYB dual color break apart FISH (CytoTest Inc, USA) was performed in all PAC and EMC cases showing MYB immunopositivity, and in MYB immunonegative de novo EMCs from non-major salivary gland sites. FISH using MAML2 dual color break apart probe (ZytoVision, Germany) and EWSR1 dual color break apart probe (CytoTest Inc, USA) was done in the index case. MYBL1 breakapart probe (Empire Genomics, USA) was used in one archival case. At least 100 non-overlapping intact nuclei were assessed manually on Zeiss Axio Imager.D2; Carl Zeiss Microscopy), and images were captured using a digital FISH imaging analysis system (Isis; MetaSystems GmbH). The presence of breakapart signals, i.e., more than two signal widths apart, in at least ten percent of tumor nuclei was considered as rearrangement.

Results

Histopathological, Immunohistochemical and Ultrastructural Features of Index Case

The biopsy (Fig. 2) showed a tumor with tubular, nested, and cribriform architecture. Some of the tubules appeared to be bilayered on hematoxylin and eosin (HE) staining. Tumor cells lining the tubules had abundant eosinophilic cytoplasm. The cribriform foci showed sharply defined spaces containing basophilic matrix surrounded by cells with abundant eosinophilic to clear cytoplasm. An occasional squamous morule was seen. IHC revealed luminal positivity for CK7, and p40 staining in abluminal cells. Beta-catenin showed membranous staining. As the features were those of an oncocytic neoplasm with focal squamoid features, oncocytic mucoepidermoid carcinoma (MEC) was considered. However, MAML2 FISH did not show rearrangement. Another possibility was EMC with oncocytic cells. The biopsy was reported as an oncocytic neoplasm, and excision was advised.

Fig. 2.

Biopsy findings: biopsy from the tumor shows predominant tubular (A) and focal cribriform and solid (B) architecture. The cribriform foci showed punched out spaces containing basophilic matrix (C) surrounded by cells with abundant eosinophilic (D) to clear (E) cytoplasm. Bilayering was evident (F). Tumor cells lining tubules had abundant eosinophilic cytoplasm (G). Squamous morules were seen (H, I). Beta-catenin showed membranous staining (J)

The resection specimen (Fig. 3) showed a tumor measuring 3.2 × 2 × 1.6 cm. Microscopic sections showed a widely infiltrative tumor composed of large cells with abundant eosinophilic cytoplasm arranged in solid nests and tubules. Some of the tumor cells had clear cytoplasm. Nuclei were round to ovoid, vesicular, with conspicuous to prominent nucleoli. Basaloid abluminal cells could be appreciated at places; these had scant cytoplasm, and small flat to ovoid hyperchromatic nuclei. However, in most areas, bilayering was not readily apparent on HE-stained sections. These features were present in approximately 70% of tumor area. Few well-formed tubules had eosinophilic material within their lumina and reduplicated basement membrane material around them. Extracellular basophilic matrix was present within sharply demarcated microcystic spaces surrounded by cells with abundant eosinophilic or clear cytoplasm, imparting a luminal cribriform appearance; typical basaloid cribriform morphology of AdCC was absent. Occasional micropapillae lined by cells with hypereosinophilic inclusion-like cytoplasm were also present. Glomeruloid structures and Paneth-like cells were not seen. Necrosis, increased mitoses, perineural invasion, and lymphovascular invasion were absent. The possible diagnoses considered were MEC, EMC, and AdCC-STE. On IHC, luminal cells were positive for CK7, p63-stained abluminal cells, and SOX10 showed diffuse positivity with abluminal accentuation, confirming the presence of a myoepithelial cell population and excluding MEC. MYB was positive, with stronger staining in the abluminal cells. Mammoglobin, NR4A3, and RAS Q61R were negative.

Fig. 3.

Final histopathology: the resected tumor has a pseudocapsule that is infiltrated (A). The tumor shows solid nests (B), tubules (C), and syncytial pattern (D). Biphasic nests show luminal cells with abundant clear cytoplasm (E); tubules are lined by columnar cells with abundant pale eosinophilic cytoplasm (F) and few pseudopapillae have cells with hypereosinophilic cytoplasm (G). Abluminal cells have scant cytoplasm and small hyperchromatic nuclei while luminal cells have round to ovoid, vesicular nucleoli with prominent nucleoli (H). Foci of stromal hyalinization are present (I). Basophilic matrix is seen within microcystic spaces surrounded by cells with abundant eosinophilic or clear cytoplasm, imparting a luminal cribriform appearance (J). CK7 highlights luminal cells (K) and p63 stains abluminal cells (L); SOX10 shows diffuse positivity (M). MYB is positive (N). FISH shows MYB (O) and EWSR1 (P) rearrangement

With a suspected diagnosis of AdCC-STE, FISH was done. Both MYB and EWSR1 FISH showed breakapart signals indicative of rearrangements. A final diagnosis of AdCC-STE was rendered. Tumor was present at the resection margins. Neck dissection yielded sixteen lymph nodes, all of which were free of tumor. The patient was planned for adjuvant radiotherapy.

Transmission electron microscopy (Fig. 4) revealed epithelial cells containing rough endoplasmic reticulum and free ribosomes, surrounded by a basal lamina. Flocculent chromatin within the nuclei showed some condensation at the nuclear membrane. Dense secretory granules of 450 to 600 nm and cytoplasmic filaments were also seen. Myoepithelial cells with more electron dense nucleus, irregular nuclear outline and cytoplasmic myofilaments were also identified. The intercellular spaces showed stellate granules corresponding to basophilic matrix seen on light microscopy. These features corroborated with those previously described in AdCC. However, mitochondria were not prominent, indicating that the luminal cells are not true oncocytes.

Fig. 4.

Ultrastructural features of index case: Elongated myoepithelial cell (m) with irregular nuclear outline and closely packed bundles of cytoplasmic myofilaments (arrow) and epithelial cell (e) surrounded by a basal lamina (*) are seen (A); epithelial cells show and dense secretory granules (arrowhead) and intermediate filaments (B, C); granular basement membrane material is seen in the extracellular space (D)

Review of Archival Cases of PAC and EMC

Fourteen cases of PAC were identified from our departmental archives, diagnosed over a period of eight years. On review of their histological features, none of them resembled AdCC-STE. MYB IHC showed immunopositivity in two cases, both of which were biopsies. MYB FISH showed breakapart signals indicative of rearrangement in one of them, confirming it to be AdCC. However, it lacked the presence of luminal hypereosinophilia and was an AdCC with classic histology having cribriform and tubular architecture with basaloid cells (Fig. 5A–D). The MYB-immunopositive FISH-negative case also did not show tubular hypereosinophilia.

Fig. 5.

Cases of PAC and EMC with MYB positivity: Archival case of PAC with cribriform architecture, foci of necrosis (A) and basaloid cells (B), immunopositive for MYB (C) and showing MYB rearrangement (D); archival case of floor of mouth EMC (E) with tubulo-trabecular (F) and solid (G) architecture, luminal cells with abundant eosinophilic cytoplasm (H), cribriform foci with basophilic matrix in pseudocystic spaces (I), focal MYB immunopositivity (J) and FISH showing normal MYB (K) and MYBL1 (L) signals

Our archives yielded 43 specimens of EMC from 36 patients. Of these, 20 tumors were located in parotid, 3 in submandibular gland, and 13 in non-major salivary gland sites. The latter included 10 cases from the oral cavity (5 in hard palate, 4 in buccal mucosa, 1 in floor of mouth), and one each from the sinonasal region, orbit and oropharynx. MYB immunostaining was performed on 35 specimens, after excluding five from the parotid, three of which were EMC ex pleomorphic adenoma, and three in which tumor tissue was exhausted. Focal MYB immunopositivity of was detected in two specimens from one patient with a floor or mouth tumor (Fig. 5). On morphology, the tumor had tubulo-trabecular and solid architecture. At places, luminal cells lining tubules had abundant eosinophilic cytoplasm in ~ 10% of tumor area. MYB and MYBL1 FISH did not show rearrangement, while EWSR1 FISH failed due to background fluorescence obscuring signals. While this could be a possible case of AdCC-STE, it could not be confirmed as such on molecular testing. MYB FISH on 11 MYB immunonegative non-major salivary gland EMC specimens did not show rearrangement.

Discussion

AdCC is a malignant epithelial neoplasm of major and minor salivary glands with propensity to occur in all seromucinous gland sites. It usually presents as a locally infiltrative tumor with frequent perineural invasion on microscopy, and a clinical predilection for local recurrence and distant metastasis, necessitating accurate diagnosis and management [5]. The biphasic nature with predominance of basaloid myoepithelial cells and variable proportion of ductal cells, coupled with the architectural patterns viz. tubular, cribriform, and solid, and the typical basophilic matrix within pseudocystic spaces imparting a Swiss cheese appearance facilitate its diagnosis, which is easily made on HE staining alone in majority of cases. However, solid pattern alone with lack of basophilic matrix, high-grade transformation, and other unusual morphologies may necessitate confirmation of the diagnosis by molecular testing. This has now come to include AdCC-STE recently described by Weinreb et al. in a series of 16 cases with predominance of hypereosinophilic luminal cells, non-major salivary gland locations, and canonical as well as novel gene fusions involving EWSR1 and FUS genes [10]. Most cases of AdCC-STE were located in the sinonasal tract (7 cases; 44%), two each were in the oral cavity, base of tongue, and external auditory canal, and the remaining were in the larynx, trachea, and nasopharynx.

The present case shares similarities with the cases described. The tumor was located in the floor of mouth. Architecture was predominantly tubular and nested, with focal solid and micropapillary patterns. Bilayering of tubules with luminal epithelial and abluminal myoepithelial cells was discernible on HE sections. Tubular hypereosinophilia was present in the form of large luminal cells with abundant eosinophilic to clear cytoplasm and uniform round nuclei with vesicular chromatin and prominent nucleoli. Luminal cribriform pattern with basophilic matrix was present; however, basaloid cribriform pattern was not seen. Reduplicated basement membrane material was seen around some of the tubules. Perineural and lymphovascular invasion were absent, similar to the previously described cases, and may be reflective of the non-parotid location of the tumor. On IHC, bilayering was evident with CK7 and p63 staining luminal and abluminal cells, respectively. p63 was negative in luminal cells showing tubular hypereosinophilia and the squamoid morules, excluding true squamous differentiation. FISH performed using dual color breakapart probes showed the presence of MYB and EWSR1 gene rearrangements, indicative of EWSR1::MYB gene fusion, first described in AdCC-STE.

On HE-stained sections, AdCC-STE is not easily recognizable as a morphological subtype of AdCC, and several benign as well as malignant neoplasms enter the differential diagnosis, including PAC, MEC, EMC, and pleomorphic adenoma (PA). PAC, like AdCC, is frequent in minor salivary gland sites. However, it displays more diverse architectural patterns, including linear cords, trabeculae, microcysts, papillary, and glomeruloid formations, apart from tubular and solid patterns [13]. Unlike AdCC, PAC is not biphasic; the tubules are unilayered, lined by intercalated duct-type luminal cells. However, their immunohistochemical profiles overlap considerably. Immunohistochemically, the main distinction between PAC and AdCC lies in the abluminal tumor cells being p63 positive and p40 negative in PAC, while those in AdCC are positive for both [14]. MYB overexpression by IHC, although sensitive, is not specific for AdCC and has been described in PAC [15]. Similarly, CD117 and smooth muscle actin expression may be seen in both. The definitive distinguishing feature, however, is the presence of alterations in PRKD genes in PAC as opposed to MYB/MYBL1 rearrangements in AdCC. Two PACs from our archives showed MYB immunopositivity; one of these showed MYB rearrangement, confirming it to be AdCC. The morphological features were more in line with AdCC than PAC, highlighting the potential for misdiagnosis on limited tissue specimens such as a biopsy. MYB/MYBL1 overexpression has been reported in AdCCs lacking MYB/MYBL1 fusions [7, 15]. The precise mechanism for this was unknown until recently when Ueda et al. showed that peri-MYB/MYBL1 rearrangements resulting in juxtaposition of super-enhancers into MYB/MYBL1 are responsible for this occurrence [16]. Our second MYB-immunopositive PAC not backed by MYB rearrangement, and possibly some reported in literature, could truly be AdCCs explained by this. However, neither of the two PAC cases reclassified as AdCC showed luminal hypereosinophilia; thus, review of archival PACs did not yield any further case of AdCC-TE.

The presence of squamoid appearing cells coupled with hypereosinophilic cells may raise the possibility of MEC, particularly the oncocytic subtype, which was the favored diagnosis on biopsy in the present case. Notably, AdCC-STE had not been described at the time of reporting of the biopsy in late 2022. MEC typically shows the presence of mucous, intermediate, and squamoid cells, p63 and p40 positivity, absent staining with calponin, SMA, S-100, and SOX10, and harbors MAML2 gene rearrangement in most cases [17–19]. The presence of biphasic tubules raises the possibility of EMC, in which the luminal, abluminal or both cell types can show oncocytic features with abundant eosinophilic to clear cytoplasm [20]. Unlike conventional AdCC in which the nuclei appear angulated and hyperchromatic, the nuclei of AdCC-STE are akin to those of EMC. The presence of clefting between tumor cell nests and stroma, as seen focally in the present case, may be indicative of AdCC-STE. Additionally, AdCC has more widely invasive borders as compared to EMC. Finally, EMC may show HRAS mutations or rearrangements of PLAG1 or HMGA2 genes in the setting of EMC ex PA, and lacks MYB/MYBL1 gene rearrangements seen in AdCC, including AdCC-STE [21]. Our reappraisal of archival EMCs revealed MYB immunopositivity in one case. Review of morphology predominantly showed trabecular architecture which is uncommon, but not undescribed in AdCC [9]. In addition, a proportion of tubular epithelial cells showed abundant eosinophilic cytoplasm. Although MYB/MYBL1 rearrangements were not demonstrable by FISH, this could possibly be a case of AdCC-STE misdiagnosed as EMC.

Although benign, PA enters the differential diagnosis of AdCC-STE, particularly due to the presence of squamoid morules. PA in minor salivary gland locations is often unencapsulated, appearing to have infiltrative borders, is more cellular, and lacks abundant myxoid matrix [22]. While a resection may show areas of typical PA, these may be absent on a biopsy, making the distinction difficult. However, the multiple phenotypes of myoepithelial cells in PA, ranging from basaloid, epithelioid, stellate, spindled, plasmacytoid, clear cell to oncocytic, point to the diagnosis [22]. Lastly, PA shows PLAG1 or HMGA2 rearrangements, and the overexpressed PLAG1 and HMGA2 proteins can be detected immunohistochemically [23]. In the present case, PA was not considered as a possibility as the tumor was definitely malignant on clinical appearance and imaging. The differential diagnosis of AdCC-STE is summarized in Table 1 [24–28].

Table 1.

Comparison of clinicopathological features of adenoid cystic carcinoma with striking tubular hypereosinophilia and its differential diagnoses

Differential diagnosis	Location	Histological features	S-100	SMA	p63	p40	CK7	CD117	Additional IHC stains	Diagnostic molecular alterations	Prognosis
Adenoid cystic carcinoma with striking tubular hypereosinophilia	Non-major salivary glands	Biphasic infiltrative tumor with luminal cribriform, tubular, solid, micropapillary patterns; luminal cells with hypereosinophilic to clear cytoplasm	Patchy +	+ / − in ME cells	+ in ME cells	+ in ME cells	+ in ductal cells	+ in ductal cells	MYB, SOX10 +	Canonical MYB/MYBL1::NFIB fusion or variant EWSR1/FUS::MYB fusion	Unknown
Classic adenoid cystic carcinoma	Major, minor salivary glands	Biphasic infiltrative tumor with cribriform, tubular or solid patterns; basaloid myoepithelial cells predominate; reduplicated basement membrane material	Patchy +	+ / − in ME cells	+ in ME cells	+ in ME cells	+ in ductal cells	+ in ductal cells	MYB, SOX10 + ; CEA, EMA + in ductal cells	MYB or MYBL1 rearrangement, most frequently MYB::NFIB fusion	Poor, with 10-year DSS of 50%
Polymorphous adenocarcinoma	> 95% in minor salivary glands	Diverse patterns: linear, trabecular, microcystic, unilayered tubules, solid; uniform cells with scant cytoplasm, bland nuclei	Diffuse strong +	− / +	Patchy +	−	Diffuse +	+ / −	Variable Mammaglobin, CEA, GFAP	PRKD gene family alterations	Excellent, with 10-year DSS > 90%
Mucoepidermoid carcinoma	Major, minor salivary glands	Solid cystic tumor with presence of mucous, intermediate and squamoid cells	−	−	+	+	+	−	SOX10-	MAML2 rearrangement	Good to intermediate, based on grade
Epithelial myoepithelial carcinoma	Major > minor salivary glands	Biphasic tumor with luminal eosinophilic ductal cells and abluminal clear ME cells	+ / − in ME cells	+ / − in ME cells	+ in ME cells	+ in ME cells	+ in ductal cells	+ / − in ductal cells	EMA + in ductal cells; RASQ61R + / −	HRAS mutations; PLAG1 or HMGA2 rearrangements	Good, with 10-year DSS of > 90%
Pleomorphic adenoma	Major and minor salivary glands	Biphasic tumor with multiple ME cell phenotypes: epithelioid, basaloid, stellate, clear, spindled, oncocytic, plasmacytoid; chondro-myxoid/fibrous stroma; squamous metaplasia	+ in ME cells	+ in ME cells	+ in ME cells	+ in ME cells	+ in ductal cells	+ / − in ductal cells	EMA + in ductal cells; SOX10, GFAP + in ME cells; PLAG1, HMGA2	PLAG1 or HMGA2 rearrangements	Excellent; with local recurrence in 2.9 to 6.7% cases

DSS disease-specific survival, IHC immunohistochemistry, ME myoepithelial, + positive, − negative

In an attempt to characterize the hypereosinophilic cells in AdCC-STE, we performed ultrastructural examination by TEM which revealed that the ductal epithelial cells do not contain mitochondria and are therefore not true oncocytes. This is supported by the homogeneous non-granular appearance of their cytoplasm. SOX10 positivity accompanied by CK7 staining and negativity for p63/p40 in these cells suggests that they have an intercalated duct/terminal duct phenotype. It may be speculated that he close proximity of these ducts in minor salivary glands to the external environment, unlike in major salivary glands, may be responsible for the proclivity of AdCC-STE for these sites.

Molecular techniques to identify the gene fusions can be broadly divided into DNA-based assays which include conventional karyotyping and FISH, and RNA-based assays like reverse-transcriptase polymerase chain reaction (RT-PCR) and RNA sequencing (RNA-Seq), either targeted or whole transcriptome. Some researchers have even developed salivary gland tumor-specific NGS panels [29]. NGS-based high-throughput assays are believed to be technically superior due to the ability to detect novel fusion breakpoints and identify novel fusion gene partners [30]. FISH and RT-PCR are, however, more widely employed as they are readily available in most diagnostic molecular pathology laboratories, even in low- and middle-income countries (LMICs). Despite being low throughput and labor intensive, they offer the advantages of lower cost and significantly shorter turn-around time. In the current case, the presence of a biphasic neoplasm with hypereosinophilic luminal cells prompted us to interrogate the tumor for MYB and EWSR1 rearrangements by FISH as these probes were readily available with us.

Conclusion

The index case highlights the expanding morphological spectrum of salivary gland tumors in general, and AdCC in particular. The presence of tubular hypereosinophilia, as opposed to the classic basaloid cribriform morphology, can make the diagnosis of AdCC-STE challenging, leading to misdiagnosis as PAC and EMC among others. Awareness of such unusual morphological subtypes of AdCC is imperative, as it has worse outcome than most salivary gland carcinomas. The presence of basophilic matrix even in the absence of basaloid myoepithelial cells and squamoid morules in a biphasic tumor with hypereosinophilic cells should raise suspicion for AdCC-STE and prompt molecular testing for confirmation, in order to avoid misdiagnosis as lower-grade malignancies or benign neoplasms, especially on biopsies. FISH can be employed to detect known gene rearrangements in such cases due to its wider accessibility, significantly shorter turn-around-time and cost-effectiveness, especially in LMICs, where NGS-based RNA-seq may not be easily accessible.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Urvashi Yadav, RM, JS, and AK. The first draft of the manuscript was written by UY and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

Funded by Department of Biotechnology, Ministry of Science and Technology, Government of India (Grant No. BT/PR27805/NER/95/1378/2018 awarded to author AK).

Data Availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Declarations

Conflict of interest

The authors have no competing interests to declare.

Ethical Approval

Approval was obtained from the Institutional Ethics Committee to conduct this study on patient samples.

Informed Consent

Consent for publication was obtained for every individual person’s data included in the study.