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Acinic cell carcinoma of the salivary gland (AciCC) is a low-grade carcinoma characterized by the overexpression of the transcription factor nuclear receptor subfamily 4 group A member 3 (NR4A3). AciCC has been the subject of a few molecular research projects. This study delves into AciCC's molecular landscape to identify additional alterations and explore their clinical implications. RNA sequencing and immunohistochemical staining for markers NR4A3/NR4A2, DOG-1, S100, and mammaglobin were utilized on 41 AciCCs and 11 secretory carcinoma (SC) samples. NR4A3 was evident in 35 AciCCs, while the residual 6 were NR4A3-negative and NR4A2-positive; SC samples were consistently NR4A3-negative. A novel fusion, PON3 exon 1- LCN1 exon 5, was detected in 9/41 (21.9%) AciCCs, exhibiting a classical histologic pattern with serous cell components growing in solid sheets alongside the intercalated duct-like component. Clinical follow-up of 39 patients over a median of 59 months revealed diverse prognostic outcomes: 34 patients exhibited no disease evidence, whereas the remaining 5 experienced poorer prognosis, involving local recurrence, lymph node, and distant metastasis, and disease-associated death, 4 of which harbored the PON3::LCN1 fusion. In addition, the HTN3::MSANTD3 fusion was recurrently identified in 7/41 AciCC cases. SC patients lacked both fusions. Immunohistochemistry uncovered differential expression of DOG-1, S100, and mammaglobin across samples, providing nuanced insights into their roles in AciCC. This study accentuates PON3::LCN1 and HTN3::MSANTD3 fusions as recurrent molecular events in AciCC, offering potential diagnostic and prognostic utility and propelling further research into targeted therapeutic strategies., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Recurrent gene fusions are common in salivary gland tumors including benign tumors, such as pleomorphic adenoma (PA) and myoepithelioma (ME). In cases where chromosomal rearrangement is identified in the pleomorphic adenoma gene 1 (PLAG1) gene, different gene partners are found. Oncocytic metaplasia, characterized by oncocytes with abundant eosinophilic granular cytoplasm and hyperchromatic nuclei, is a well-known phenomenon in salivary gland neoplasms. However, the pure oncocytic variant of PA/ME showed PLAG1 gene rearrangements involving various gene partners at the molecular level, without any recurrent fusion being found. Our study includes 20 cases of PA/ME, with 11 females and 9 males. The age of patients ranged from 37 to 96 years, with a median age of 62.8 years. Most tumors originate from the parotid gland. The median size of the tumor was 26.5 mm (range: 13 to 60 mm). Among the 20 cases, 14 were a pure oncocytic variant of PA/ME, whereas 6 cases showed focal oncocytic or oncocytic-like aspects. Molecular studies on 20 cases of PA/ME were conducted. A novel recurrent ZBTB47-AS1::PLAG1 fusion was identified in 6 of 12 cases with pure oncocytic metaplasia, whereas the other cases had PLAG1 gene fusion with different gene partners. The transcriptomic analysis of the cases harboring ZBTB47-AS1::PLAG1 fusion demonstrated that these tumors have a distinct molecular profile from conventional PA/ME. This study reveals a unique subset in the oncocytic PA/ME spectrum characterized by pure oncocytic morphology with larger oncocytic cells and recurrent ZBTB47-AS1::PLAG1 fusion. It also highlights the transcriptomic distinctness of salivary gland adenomas with pure oncocytic metaplasia in the spectrum of salivary gland neoplasms. Further studies are needed to better understand the oncocytic variant of PA/ME and to determine the true nature of oncocytic cells in PA/ME., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Keratocystoma is a rare salivary gland lesion that has been reported primarily in children and young adults. Because of a scarcity of reported cases, very little is known about it, including its molecular underpinnings, biological potential, and histologic spectrum. Purported to be a benign neoplasm, keratocystoma bears a striking histologic resemblance to benign lesions like metaplastic Warthin tumor on one end of the spectrum and squamous cell carcinoma on the other end. This overlap can cause diagnostic confusion, and it raises questions about the boundaries and definition of keratocystoma as an entity. This study seeks to utilize molecular tools to evaluate the pathogenesis of keratocystoma as well as its relationship with its histologic mimics. On the basis of targeted RNA sequencing (RNA-seq) results on a sentinel case, RUNX2 break-apart fluorescence in situ hybridization (FISH) was successfully performed on 4 cases diagnosed as keratocystoma, as well as 13 cases originally diagnosed as tumors that morphologically resemble keratocystoma: 6 primary squamous cell carcinomas, 3 metaplastic/dysplastic Warthin tumors, 2 atypical squamous cysts, 1 proliferating trichilemmal tumor, and 1 cystadenoma. RNA-seq and/or reverse transcriptase-PCR were attempted on all FISH-positive cases. Seven cases were positive for RUNX2 rearrangement, including 3 of 4 tumors originally called keratocystoma, 2 of 2 called atypical squamous cyst, 1 of 1 called proliferating trichilemmal tumor, and 1 of 6 called squamous cell carcinoma. RNA-seq and/or reverse transcriptase-PCR identified IRF2BP2::RUNX2 in 6 of 7 cases; for the remaining case, the partner remains unknown. The cases positive for RUNX2 rearrangement arose in the parotid glands of 4 females and 3 males, ranging from 8 to 63 years old (mean, 25.4 years; median, 15 years). The RUNX2 -rearranged cases had a consistent histologic appearance: variably sized cysts lined by keratinizing squamous epithelium, plus scattered irregular squamous nests, with essentially no cellular atypia or mitotic activity. The background was fibrotic, often with patchy chronic inflammation and/or giant cell reaction. One case originally called squamous cell carcinoma was virtually identical to the other cases, except for a single focus of small nerve invasion. The FISH-negative case that was originally called keratocystoma had focal cuboidal and mucinous epithelium, which was not found in any FISH-positive cases. The tumors with RUNX2 rearrangement were all treated with surgery only, and for the 5 patients with follow-up, there were no recurrences or metastases (1 to 120 months), even for the case with perineural invasion. Our findings solidify that keratocystoma is a cystic neoplastic entity, one which appears to consistently harbor RUNX2 rearrangements, particularly IRF2BP2::RUNX2 . Having a diagnostic genetic marker now allows for a complete understanding of this rare tumor. They arise in the parotid gland and affect a wide age range. Keratocystoma has a consistent morphologic appearance, which includes large squamous-lined cysts that mimic benign processes like metaplastic Warthin tumor and also small, irregular nests that mimic squamous cell carcinoma. Indeed, RUNX2 analysis has considerable promise for resolving these differential diagnoses. Given that one RUNX2 -rearranged tumor had focal perineural invasion, it is unclear whether that finding is within the spectrum of keratocystoma or whether it could represent malignant transformation. Most important, all RUNX2 -rearranged cases behaved in a benign manner., Competing Interests: Conflicts of Interest and Source of Funding: Supported by Jane B. and Edwin P. Jenevein M.D Endowment for Pathology at UT Southwestern Medical Center. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Multiple 3-tiered grading systems exist for mucoepidermoid carcinoma (MEC), leading to controversial results on the frequency and prognostic values of each grade. We aimed to identify prognostic histologic factors and to evaluate grading schemes in this retrospective study of 262 resected primary head and neck MECs. The rate of nodal metastasis was 8.4%. Large tumor size, tumor fibrosis, infiltrative border, lymphovascular invasion, perineural invasion, atypical mitosis, mitotic index (MI) ≥4/2 mm 2 (4/10 HPFs), necrosis, and pT4 stage were associated with increased risk of nodal metastasis. The 5-year recurrence-free survival (RFS) was 95%. Significant prognostic factors for RFS included infiltrative border, tumor-associated lymphoid stroma, architectural patterns (macrocystic, microcystic, and noncystic), anaplasia, atypical mitosis, MI, necrosis, lymphovascular invasion, margin, pT stage, and tumor size. Nuclear anaplasia, high mitotic rate, and ≥25% microcystic component were significant independent prognostic factors on multivariate survival analysis. There was no significant difference between low-grade (LG) and intermediate-grade (IG) MECs in terms of risk of nodal metastasis and outcomes using all 4 known grading systems. Rather, high-grade MEC was consistently associated with an increased risk of nodal metastasis at presentation and decreased RFS and distant metastasis-free survival (DMFS) compared with the LG/IG MECs. We therefore recommend simplifying MEC grading to a 2-tiered grading scheme using MI and/or tumor necrosis. Using a 2-tiered grading, high-grade histology independently predict RFS, and is associated with a 25% risk of nodal metastasis, a 5-year RFS of 76%, and a 5-year DMFS of 76%, whereas LG MEC has a nodal metastasis rate of 7.0%, 5-year RFS of 97% and 5-year DMFS of 99%., Competing Interests: Conflicts of Interest and Source of Funding: Supported in part by the Cancer Center Support Grant of the National Institutes of Health/National Cancer Institute under award number P30CA008748. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Adenocarcinoma, not otherwise specified (NOS) is a heterogenous group of salivary gland tumors that likely contains distinct tumors that have not yet been characterized. Indeed, in recent years, cases previously diagnosed as adenocarcinoma, NOS have been recategorized into novel tumor designations such as secretory carcinoma, microsecretory adenocarcinoma, and sclerosing microcystic adenocarcinoma. We sought to describe a distinctive, hitherto-undescribed salivary gland tumor encountered in the authors' practices. Cases were pulled from the surgical pathology archives of the authors' institutions. Histologic, immunohistochemical, and clinical findings were tabulated, and targeted next-generation sequencing was performed on all cases. Nine cases were identified, arising in 8 women and 1 man ranging from 45 to 74 years (mean, 56.7 y). Seven tumors (78%) arose in the sublingual gland, while 2 (22%) arose in the submandibular gland. The cases shared a distinctive morphologic appearance. They were biphasic, with ducts scattered among a predominant polygonal cell with round nuclei, prominent nucleoli, and pale eosinophilic cytoplasm. These cells were arranged as trabeculae and palisaded as pseudorosettes around hyalinized stroma and vessels, resembling a neuroendocrine tumor. Four of the cases were well-circumscribed, while the remaining 5 showed infiltrative growth including perineural invasion in 2 (22%) and lymphovascular invasion in 1 (11%). Mitotic rates were low (mean, 2.2/10 HPFs); necrosis was absent. By immunohistochemistry, the predominant cell type was strongly positive for CD56 (9 of 9) and variably positive for pan-cytokeratin (AE1/AE3) (7 of 9) with patchy S100 (4 of 9), but negative for synaptophysin (0 of 9) and chromogranin (0 of 9), while the ducts were strongly positive for pan-cytokeratin (AE1/AE3) (9 of 9) and CK5/6 (7 of 7). Next-generation sequencing did not reveal any fusions or obvious driver mutations. All cases were resected surgically, with external beam radiation also done in 1 case. Follow-up was available in 8 cases; there were no metastases or recurrences after 4 to 160 months (mean, 53.1 mo). A dual population of scattered ducts with a predominance of CD56-positive neuroendocrine-like cells characterizes a unique salivary gland tumor which is often encountered in the sublingual glands of women, for which we propose the term "palisading adenocarcinoma." Although the tumor was biphasic and had a neuroendocrine-like appearance, it lacked convincing immunohistochemical evidence of myoepithelial or neuroendocrine differentiation. Although a subset showed unequivocally invasive growth, this tumor appears to behave in an indolent manner. Moving forward, recognition of palisading adenocarcinoma and its separation from other salivary adenocarcinomas, NOS will facilitate a better understanding of the characteristics of this previously unrecognized tumor., Competing Interests: Conflicts of Interest and Source of Funding: Supported by Jane B. and Edwin P. Jenevein M.D Endowment for Pathology at UT Southwestern Medical Center. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

The aim of this study was to evaluate the clinicopathologic features, molecular characteristics, treatment strategy, and prognosis of nasopharyngeal hyalinizing clear cell carcinoma (HCCC). Retrospective observational case series. Institutional pathology records between 2006 and 2022 were searched for all cases of nasopharyngeal HCCC. We included 10 male and 16 female patients aged 30 to 82 years (median: 60.5 y, mean: 54.6 y). The most common symptoms were blood-stained rhinorrhea and nasal obstruction. Tumors most often involved the lateral wall of the nasopharynx, followed by the superior posterior wall. Microscopically, all tumor cells were arranged in sheets, nests, cords, and single cells in a hyaline/myxoid/fibrous stroma. The tumor cells were polygonal, with or without distinct cell borders, and displayed abundant clear-to-eosinophilic cytoplasm. All 26 cases were positive for pancytokeratin, CK7, p40, and p63 but negative for myoepithelial differentiation markers. Ki-67 labeling was low and ranged from 1% to 10%. All 26 cases demonstrated EWSR1 and EWSR1-ATF1 rearrangements, and no case demonstrated MAML2 rearrangement. Complete follow-up data were available for 23 patients: 14 patients underwent endoscopic surgery alone, 5 underwent radiation therapy followed by endoscopic surgery, 3 underwent radiation therapy followed by biopsy, and 1 underwent cisplatin chemotherapy before endoscopic surgery. Clinical follow-up ranged from 6 to 195 months; 13 patients (56.5%) were alive without tumor, 5 patients (21.7%) died of disease, 5 patients (21.7%) survived with tumor. HCCCs of the nasopharynx are rare tumors. The definitive diagnosis depends on histopathology, immunohistochemistry, and molecular studies. The optimal treatment for patients with nasopharyngeal HCCC is wide local excision. Radiation and chemotherapy might be good options for managing locally advanced cases. Nasopharyngeal HCCC is less indolent than previously thought. Tumor stage and the choice of treatment are key factors affecting the prognosis of nasopharyngeal HCCC patients., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Salivary gland secretory carcinoma (SC), previously mammary analog SC, is a low-grade malignancy characterized by well-defined morphology and an immunohistochemical and genetic profile identical to SC of the breast. Translocation t(12;15)(p13;q25) resulting in the ETV6 :: NTRK3 gene fusion is a characteristic feature of SC along with S100 protein and mammaglobin immunopositivity. The spectrum of genetic alterations for SC continues to evolve. The aim of this retrospective study was to collect data of salivary gland SCs and to correlate their histologic, immunohistochemical, and molecular genetic data with clinical behavior and long-term follow-up. In this large retrospective study, we aimed to establish a histologic grading scheme and scoring system. A total of 215 cases of salivary gland SCs diagnosed between 1994 and 2021 were obtained from the tumor registries of the authors. Eighty cases were originally diagnosed as something other than SC, most frequently acinic cell carcinoma. Lymph node metastases were identified in 17.1% (20/117 cases with available data), with distant metastasis in 5.1% (6/117). Disease recurrence was seen in 15% (n=17/113 cases with available data). The molecular genetic profile showed ETV6 :: NTRK3 gene fusion in 95.4%, including 1 case with a dual fusion of ETV6 :: NTRK3 and MYB :: SMR3B . Less frequent fusion transcripts included ETV6 :: RET (n=12) and VIM :: RET (n=1). A 3-tiered grading scheme using 6 pathologic parameters (prevailing architecture, pleomorphism, tumor necrosis, perineural invasion (PNI), lymphovascular invasion (LVI), and mitotic count and/or Ki-67 labeling index) was applied. Grade 1 histology was observed in 44.7% (n=96), grade 2 in 41.9% (n=90), and grade 3 in 13.5% (n=29) of cases. Compared with low-grade and intermediate-grade SC, high-grade tumors were associated with a solid architecture, more prominent hyalinization, infiltrative tumor borders, nuclear pleomorphism, presence of PNI and/or LVI, and Ki-67 proliferative index >30%. High-grade transformation, a subset of grade 2 or 3 tumors, seen in 8.8% (n=19), was defined as an abrupt transformation of conventional SC into high-grade morphology, sheet-like growth, and a tumor lacking distinctive features of SC. Both overall survival and disease-free survival (5 and 10 y) were negatively affected by tumor grade, stage, and TNM status (each P <0.0001). SC is a low-grade malignancy with predominantly solid-microcystic growth patterns, driven by a gene fusion, most commonly ETV6 :: NTRK3 . There is a low risk for local recurrence and a good overall long-term survival, with a low risk for distant metastasis but a higher risk for locoregional lymph node metastasis. The presence of tumor necrosis, hyalinization, PNI and/or LVI, and positive resection margins correlate with higher tumor grade, less favorable prognosis, and increased mortality. The statistical results allowed us to design a 3-tiered grading system for salivary SC., Competing Interests: Conflicts of Interest and Source of Funding: This study was in part supported by study grant SVV 260539 from the Ministry of Education, Czech Republic, the Cooperation Program, research area SURG, the project National Institute for Cancer Research—NICR (Programme EXCELES, ID Project No. LX22NPO5102). Funded by the European Union—Next Generation EU and the Finnish Cancer Society, Finska Läkaresällskapet, and Maritza and Reino Salonen Foundation, Finland. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

The classification of salivary gland tumors is ever-evolving with new variants of tumors being described every year. Next-generation sequencing panels have helped to prove and disprove prior assumptions about tumors' relationships to one another, and have helped refine this classification. Adenoid cystic carcinoma (AdCC) is one of the most common salivary gland malignancies and occurs at all major and minor salivary gland and seromucous gland sites. Most AdCC are predominantly myoepithelial and basaloid with variable cribriform, tubular, and solid growth. The luminal tubular elements are often less conspicuous. AdCC has largely been characterized by canonical MYB fusions, with MYB::NFIB and rarer MYBL1::NFIB. Anecdotal cases of AdCC, mostly in nonmajor salivary gland sites, have been noted to have unusual patterns, including squamous differentiation and macrocystic growth. Recently, this has led to the recognition of a subtype termed "metatypical adenoid cystic carcinoma." Another unusual histology that we have seen with a wide range of architecture, is striking tubular hypereosinophilia. The hypereosinophilia and luminal cell prominence is in stark contrast to the vast majority of AdCC that are basaloid and myoepithelial predominant. A total of 16 cases with tubular hypereosinophilia were collected, forming morular, solid, micropapillary, and glomeruloid growth, and occasionally having rhabdoid or Paneth-like cells. They were subjected to molecular profiling demonstrating canonical MYB::NFIB (5 cases) and MYBL1::NFIB (2 cases), as well as noncanonical EWSR1::MYB (2 cases) and FUS::MYB (1 case). The remaining 6 cases had either no fusion (3 cases) or failed sequencing (3 cases). All cases were present in nonmajor salivary gland sites, with seromucous glands being the most common. These include sinonasal tract (7 cases), laryngotracheal (2 cases), external auditory canal (2 cases), nasopharynx (1 case), base of tongue (2 cases), palate (1 case), and floor of mouth (1 case). A tissue microarray of 102 conventional AdCC, including many in major salivary gland sites was examined for EWSR1 and FUS by fluorescence in situ hybridization and showed that these novel fusions were isolated to this histology and nonmajor salivary gland location. In summary, complex and striking tubular hypereosinophilia and diverse architectures are present within the spectrum of AdCC, particularly in seromucous gland sites, and may show variant EWSR1/FUS::MYB fusions., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

The landscape of salivary gland carcinomas is ever-changing, with a growing list of new tumors and newly elucidated variants of well-known tumor entities. The routine use of next-generation sequencing has been instrumental in identifying novel fusions and tumor entities, which has helped bring the classification to a more objective and evidenced-based model. However, morphology remains critical in assessing the validity of these novel molecular findings, and most importantly, in assessing which of these findings will have an impact on the prognosis and treatment decisions for patients. The recognition of microsecretory adenocarcinoma (MSA) as a distinct low-grade malignancy of salivary glands, underpinned by MEF2C::SS18 , and a single possibly related case of SS18::ZBTB7A , recently expanded this growing list of distinctive tumors. It was not until now, however, that the morphology of the latter case was known to be unique and reproducible. The authors have now seen 4 of these distinctive tumors that show a combination of distinctive oncocytic cells forming compact glandular growth as well as amphophilic cells forming tubular growth, and suggest the appellation "microcribriform adenocarcinoma" (MCA). So far, these tumors appear to preferentially occur in nonoral sites (2 parotid, 1 submandibular gland, and 1 bronchial seromucous glands). By immunohistochemistry, they express S100 and SOX-10 with focal outer myoepithelial cells marked by circumferential p63, p40, and smooth muscle actin staining around some of the nests and tubules. The tumors show infiltrative growth within a hyalinized and myxoid stroma. Cytologically, they appear generally low grade, similar to MSA. The morphologic and molecular uniformity of these 4 microcribriform adenocarcinoma cases warrants their recognition, and while related to MSA, they are sufficiently different to be classified as a distinct tumor. So far, in limited follow-up, these tumors appear to be relatively indolent., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Breast adenoid cystic carcinoma (AdCC) has overlapping features with basal-like triple-negative breast carcinoma (TNBC), yet carries a more favorable prognosis, and accurate diagnosis is critical. Like salivary gland AdCC, breast AdCC demonstrates recurrent alterations in the MYB gene. Novel chromogenic RNA in situ hybridization (ISH) for MYB has emerged as sensitive and specific for salivary gland AdCC. Here, we evaluate MYB RNA ISH in invasive ductal carcinomas (IDCs) including basal-like TNBC, and in the histologic mimics ductal carcinoma in situ (DCIS) and collagenous spherulosis. MYB RNA ISH was also performed on previously constructed tissue microarrays containing 78 evaluable IDC, including 30 basal-like TNBC (EGFR+ and/or CK5/6+), 19 luminal A (ER+/HER-2-), 12 HER-2+ (ER-/HER-2+), 11 non-basal-like TNBC, and 6 luminal B (ER+/HER-2+). MYB RNA ISH overexpression was seen in 100% (n=18/18) of primary breast AdCC and 10% (n=8/78) of IDC (P<0.0001). MYB RNA ISH was overexpressed in 37% (n=7/19) of luminal A and 8% (n=1/12) of HER-2+ IDC, and in no cases of TNBC or luminal B IDC. The majority (67%, n=8/12) of DCIS and all (n=7) cases of collagenous spherulosis demonstrated overexpression of MYB RNA. MYB gene rearrangement was detected in 67% (n=4/6) evaluable AdCC. Although MYB RNA ISH overexpression cannot be used to distinguish between cribriform DCIS or collagenous spherulosis and AdCC, MYB RNA ISH is absent in basal-like TNBC and rare in ER+ or HER-2+ IDC. MYB RNA ISH could be a useful, sensitive, and rapid diagnostic adjunct in the workup of a triple-negative carcinoma in the breast., Competing Interests: Conflicts of Interest and Source of Funding: Supported by the Center of Excellence Award from the Avon Foundation for Women (A.C-M.). The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Adenoid cystic carcinoma is a malignant salivary gland neoplasm, commonly involving the major and minor salivary glands. Adenoid cystic carcinoma arising in the skull base region is considerably less common and is characterized by aggressive clinical behavior, perineural invasion, and intracranial extension. Classically, these tumors are composed of ductal and myoepithelial cells, arranged as tubules and cribriform structures, as well as in a solid pattern when higher in grade. The distinctive molecular findings in this tumor are the gene fusions involving the MYB/MYBL1 and NFIB genes. Squamous differentiation, trabecular, and macrocystic growth patterns are exceedingly rare in these tumors and when present can cause significant diagnostic challenges. Squamous differentiation, in particular, is considered by many to be an exclusion criterion for adenoid cystic carcinoma outside of cases with high-grade transformation. In addition, a similar-appearing tumor with squamous differentiation, namely human papillomavirus-related multiphenotypic sinonasal carcinoma, has recently been defined, further complicating this differential diagnosis. Recently, we have come across 3 cases of adenoid cystic carcinomas involving the sinonasal tract and skull base having extensive interconnecting trabecular growth, macrocysts, and squamous differentiation, yet demonstrating the signature fusions involving MYB-NFIB and MYBL1-NFIB by RNA sequencing. In this article, we describe the clinical, histomorphologic, and imaging findings of these cases and propose the appellation "metatypical adenoid cystic carcinoma" for this uncommon variant morphology., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Most of salivary gland neoplasms (benign and malignant) are characterized by recurrent gene fusions. Pleomorphic adenoma (PA), the most frequent salivary gland tumor, is driven by chromosomal rearrangements involving PLAG1 mapped to 8q12 and HMGA2 mapped to 12q13-15 in most cases. Multiple fusion partners have been identified including CTNNB1, FGFR1, LIFR, CHCHD7 and TCEA for PLAG1 fusions and NFIB, WIF1 and FHIT for HMGA2 fusions. To date, no data exist on the morphology of the few reported HMGA2-WIF1-rearranged PAs. We present 28 major salivary gland adenomas displaying distinctive trabecular and canalicular morphology associated with recurrent genotype. Patients were 15 females and 13 males aged 43 to 87 (median: 65). All tumors originated from the parotid. Their size range was 1 to 4 cm (mean: 2.3). Histologically, all tumors showed elongated or columnar cells arranged into bilayered to multilayered communicating and branching strands and trabeculae in a manner similar to canalicular adenoma of minor salivary glands or trabecular myoepithelioma with variable solid confluent intercalated duct-like areas. Fifteen tumors were exclusively canalicular/trabecular while 13 had intermingled or well-demarcated conventional (chondromyxoid) PA component comprising 5 to >50% of the tumor. The monomorphic areas expressed uniformly CK7 (28/28), vimentin (21/21), S100 (24/24), SOX10 (16/17) and variably p63 (8/21) and mammaglobin (6/16) but were negative with p40 (0/24), smooth muscle actin (0/24) and MUC4 (0/16). Targeted RNA sequencing revealed HMGA2 fusions in 14/16 (87%) assessable cases. Fusion partner was WIF1 (12), RPSAP52 (1) and HELB (1). Separate testing of the 2 components in 1 hybrid tumor showed same HMGA2/WIF1 fusion. HMGA2 immunohistochemistry was homogeneously positive in all cases including the 2 fusion-negative cases. A control cohort of 12 genuine canalicular adenomas revealed no HMGA2 fusions (0/4) and lacked HMGA2 immunoreactivity (0/12). This study highlights a distinctive variant in the spectrum of PA characterized by prominent trabecular and canalicular adenoma-like morphology. Our data confirm that canalicular adenomas in major salivary glands (either monomorphic or part of hybrid tumors) are distinct from canalicular adenoma of minor salivary glands. Their uniform genotype irrespective of presence or absence of a conventional PA component argues for classifying those tumors lacking a conventional PA component as "monomorphic variants of PA" rather than canalicular/basal cell adenomas, intercalated duct adenoma, trabecular myoepithelioma or true hybrid tumors., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Aims: Secretory carcinoma (SC) is characterized by ETV6 rearrangements, most often ETV6-NTRK3 fusion. Given its histologic overlap with other salivary gland tumors (SGTs), SCs can be difficult to diagnose without genetic confirmation. A recently developed pan-TRK (tropomyosin receptor kinase) antibody shows promise for identifying tumors with NTRK (neurotrophic tyrosine kinase receptor 3) fusions. The aim of this study was to evaluate the utility of pan-TRK immunohistochemistry in distinguishing SCs from mimics and selecting patients eligible for TRK inhibitor clinical trials. We examined whole-tissue sections from 111 SGTs with molecular characterization, including 26 SCs (23 with ETV6-NTRK3 fusion and 3 with ETV6-RET fusion detected by ligation-dependent reverse transcription-polymerase chain reaction, next-generation sequencing and 85 non-SC SGTs (no ETV6-NTRK3 fusion). Immunohistochemistry was performed with a pan-TRK rabbit monoclonal antibody. When any pan-TRK staining (nuclear or cytoplasmic with any staining intensity) was considered to indicate positivity, 22 of 23 SCs with ETV6-NTRK3 fusion (95.7%) and 33 of 85 non-SC (38.8%) salivary neoplasms were positive, mainly basal cell adenoma, pleomorphic adenomas, adenoid cystic carcinomas, and epithelial-myoepithelial carcinomas. All SCs with ETV6-RET fusion were entirely negative. When only nuclear pan-TRK staining with any staining intensity was considered positive, 18 of 23 SCs with ETV6-NTRK3 fusion (78.3%) were positive, 11 among them with diffuse staining (>30% of cells). All non-SCs and SCs with ETV6-RET fusion were entirely negative. In comparison to molecular analysis (ligation-dependent reverse transcription-polymerase chain reaction, next-generation sequencing), nuclear pan-TRK IHC has a sensitivity of 78.3% and a specificity of 100% for diagnosing SCs with ETV6-NTRK3 fusion, 69% and 100% for SCs (all fusions). Pan-TRK is a reasonable screening test for diagnosing SCs among SGTs when taking only nuclear staining into account. Although pan-TRK expression is not entirely sensitive for SCs, nuclear staining is highly specific for SCs with ETV6-NTRK3 fusion. The lack of pan-TRK immunoreactivity in a subset of SCs is suggestive of atypical exons 4 to 14 or exons 5 to 14 ETV6-NTRK3 fusion or non-NTRK alternative fusion partners such as ETV6-RET. Pan-TRK staining can serve as a strong diagnostic marker to distinguish SC from it mimics and to select patients eligible for TRK inhibitor clinical trials., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Mucin-producing salivary adenocarcinomas were historically divided into separate colloid carcinoma, papillary cystadenocarcinoma, and signet ring cell carcinoma diagnoses based on histologic pattern, but have recently been grouped together in the adenocarcinoma not otherwise specified category. It is currently unclear if these tumors represent 1 or more distinct entities and how they are related to well-circumscribed papillary mucinous lesions with recurrent AKT1 E17K mutations that were recently described as salivary intraductal papillary mucinous neoplasm. Here, we sought to evaluate the clinicopathologic and molecular features of salivary mucinous adenocarcinomas to clarify their classification. We identified 17 invasive mucin-producing salivary adenocarcinomas, 10 with a single histologic pattern, and 7 with mixed patterns. While most tumors demonstrated papillary growth (n=15), it was frequently intermixed with colloid (n=6) and signet ring (n=3) architecture with obvious transitions between patterns. All were cytokeratin 7 positive (100%) and cytokeratin 20 negative (0%). Next-generation sequencing performed on a subset demonstrated recurrent AKT1 E17K mutations in 8 cases (100%) and TP53 alterations in 7 cases (88%). Of 12 cases with clinical follow-up (median: 17 mo), 4 developed cervical lymph node metastases, all of which had colloid or signet ring components. Overall, overlapping histologic and immunohistochemical features coupled with recurrent AKT1 E17K mutations across patterns suggests that mucin-producing salivary adenocarcinomas represent a histologically diverse single entity that is closely related to tumors described as salivary intraductal papillary mucinous neoplasm. We propose a unified mucinous adenocarcinoma category subdivided into papillary, colloid, signet ring, and mixed subtypes to facilitate better recognition and classification of these tumors., Competing Interests: Conflicts of Interest and Source of Funding: Supported in part by the Jane B. and Edwin P. Jenevein, MD Endowment for Pathology at UT Southwestern Medical Center. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The majority of salivary gland carcinomas are characterized by recurrent gene fusions that proved highly valuable diagnostically, but only rarely of therapeutic impact. Most of these fusion-positive carcinomas belong to the low-grade or intermediate-grade biological category. To date, only 5 cases of salivary gland carcinomas carrying an oncogenic ALK fusion have been reported in 4 recent studies, but their phenotypic spectrum and their nosological classification remain uncharacterized. We herein describe in detail the clinicopathologic and molecular features of 4 ALK-fusion-positive salivary carcinomas and review previously reported cases to assess if they could be classified into a defined World Health Organization (WHO) category. Patients were 3 men and 1 woman aged from 67 to 79 years (median: 70 y). All tumors originated in the parotid gland. Their size ranged from 1.1 to 3 cm (mean, 2 cm). Three tumors were de novo high-grade salivary duct carcinomas (SDCs) and 1 was a low-grade intercalated-type intraductal carcinoma. Histologically, high-grade tumors were predominantly solid, composed of intimately admixed basal (CK5+, androgen-) and luminal (CK5-, androgen+) components. The remarkable basal component showed squamoid basophilic pattern imparting an adenosquamous-like appearance in all cases. Conventional apocrine intraductal high-grade carcinoma was noted in 1 case. Prominent intraductal growth of the solid basal component (highlighted by p63 staining) was seen in all cases. The tumor cells expressed CK7 (3/3), mammaglobin (3/3, 1 focal), GATA3 (3/3, 1 focal), variably CK5 (3/3), and focally the androgen receptor (1/3), but lacked expression of HER2/neu, SOX10, MUC4, TTF1, S100, and Napsin A. The low-grade tumor showed classic histologic and immunophenotypic features of intercalated-type noninvasive intraductal carcinoma. Molecular profiling showed rearrangements involving exon 20 of ALK in all cases, confirmed by ALK immunohistochemistry (IHC and FISH). The fusion partner was EML4 (n=2) and STRN (n=1) in high-grade tumors and EML4 in the intraductal carcinoma. Two patients with high-grade tumors developed progressive disease (1 died at 9 mo; 1 alive under palliative therapy at 5 mo). This series and a review of 5 published cases indicate that ALK rearrangements characterize 2 distinct subsets of salivary gland carcinomas in the spectrum of high-grade androgen-poor, basal-like SDC (total reported: 5 cases) and low-grade intercalated-type intraductal carcinomas (4 cases). Given the therapeutic relevance of ALK fusions, inclusion of ALK IHC in any atypical-looking or androgen-poor SDC and in high-grade adenocarcinoma-not otherwise specified is recommended. Absence of aberrant ALK expression in genetically characterized secretory (n=15) and intraductal (n=9) carcinomas lacking ALK fusions underlines the value of ALK IHC as a diagnostic screening method for identifying potential cases., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Mucoepidermoid carcinoma (MEC) is generally characterized by an admixture of mucous, epidermoid and intermediate type cells. Numerous variants morphologies are described and defined by stromal and/or cytoplasmic tinctorial characteristics. We now report 11 cases of MEC with serous acinar differentiation, reflecting a distal intercalated duct/acinar phenotype, which we designate as mucoacinar carcinomas. Seven patients were female while 4 were male with a mean age of 55 years (range: 21 to 72 y). Ten cases were from the parotid while 1 was from the submandibular gland. Mean size of the tumors was 1.8 cm (range: 0.7 to 4.5 cm). Three cases were low grade, 7 were intermediate grade, and 1 was high grade. Low to intermediate grade cases demonstrated prominent clear to vacuolated cells with focal serous acinar differentiation. The high-grade case showed a distinctive scattering of acinar cells interspersed between epidermoid cells. Periodic acid Schiff after diastase (9/9), SOX-10 (9/9), and DOG-1 (9/10) highlighted the acinar component. Six of 7 cases showed a focal acinar predominant NR4A3 expression. MAML2 fluorescence in situ hybridization was positive in all cases, in both acinar and mucoepidermoid components. Two cases tested by next generation sequencing showed standard CRTC1-MAML2 fusions. MSANTD3 and NR4A3 fluorescence in situ hybridization on the other hand were negative. Evidence thus suggests that mucoacinar carcinoma represents an acinar variant morphology in MEC, rather than a true MEC-acinic cell carcinoma hybrid, or collision tumor. The acinar differentiation, SOX-10, DOG-1, and even focal NR4A3 reactivity may thus be diagnostic pitfalls., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article, (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Epithelial-myoepithelial carcinoma (EMC) is a rare salivary gland cancer characterized by biphasic tubular structures composed of inner ductal and outer clear myoepithelial cells. Because of its histologic variety and overlap of histologic features with other salivary gland tumors, there are broad differential diagnoses. The HRAS Q61R mutation has been reported to be frequent in and specific to EMC. We evaluated the usefulness of RAS Q61R mutant-specific immunohistochemical (IHC) staining for detecting this genetic alteration in EMC. We investigated 83 EMC cases and 66 cases of salivary gland tumors with an EMC-like component, including pleomorphic adenoma, adenoid cystic carcinoma, basal cell adenoma/adenocarcinoma, and myoepithelial carcinoma. Sanger sequencing was performed for HRAS, KRAS, and NRAS. The diffuse and membranous/cytoplasmic RAS Q61R IHC expression was observed in 65% of EMC cases, in which all cases harbored the HRAS Q61R mutation. IHC-positive cases were present only in de novo EMCs (54/76 cases, 71%) but not in EMCs ex pleomorphic adenoma. The immunoreactivity was almost always restricted to the myoepithelial cells. Conversely, all EMC cases lacking the HRAS Q61R mutation were negative on IHC. In addition, only 3% of EMC-like tumors showed the abovementioned immunopositivity. None of the cases examined carried KRAS or NRAS mutations. IHC for RAS Q61R is highly sensitive and specific for detecting the HRAS Q61R mutation in EMC. Since significant immunopositivity was almost exclusively identified in nearly two thirds of EMCs but seldom in the histologic mimics, the IHC of RAS Q61R is a useful tool for diagnosing EMC in general pathology laboratories., Competing Interests: Conflicts of Interest and Source of Funding: Supported by NIH/NHS 1P01CA240239-01 (W.C.F., P.M.S.). The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The classification of salivary gland carcinomas has become increasingly specific over the last decade with the definition of new tumor types, documentation of novel molecular and immunohistochemical findings, and development of more refined diagnostic criteria. In this setting, it is unclear how many salivary tumors still cannot be easily categorized-and whether such tumors represent undifferentiated malignancies or include additional definable entities. Relying largely on current classification schemes and contemporary immunohistochemical panels, we reassessed salivary tumors previously diagnosed as adenocarcinoma, not otherwise specified (ACA NOS) from 2 large academic medical centers. Fifty-seven ACA NOS (72%) could be reclassified as more specific entities including 31 salivary duct carcinomas (39%), 7 polymorphous adenocarcinomas (9%), 5 epithelial-myoepithelial carcinomas (6%), 4 myoepithelial carcinomas (5%), 4 secretory carcinomas (5%), 1 acinic cell carcinoma (1%), 1 basal cell adenocarcinoma (1%), 1 intraductal carcinoma (1%), and 1 clear cell carcinoma (1%) as well as 2 metastatic squamous cell carcinomas (3%). Of reclassified cases, 21 (37%) represented variant histologies within these categories. ACA NOS comprised 11% of salivary malignancies before reclassification, but only 4% after reclassification. The remaining 22 ACA NOS demonstrated heterogeneous features, with an association between histologic grade and clinical outcome. In effect, ACA NOS is becoming a bygone entity as modern classification schemes and ancillary techniques now permit more specific typing of a majority of these tumors, potentially facilitating more specific prognostication and treatment. Additional distinctive entities such as mucinous adenocarcinoma may still be definable within the ACA NOS category., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Sialadenoma papilliferum (SP) is a rare benign tumor of the salivary glands, and only 3 unequivocal cases of SP arising in the bronchus have been reported. We herein describe the histomorphologic and molecular features of 4 bronchial SP cases and discuss the differential diagnosis of this entity and the relationship with its clinicopathologic mimics, in particular, glandular papilloma and mixed squamous cell and glandular papilloma (GP/MP). We encountered 2 male and 2 female patients with bronchial SP (mean: 66.8 y old). All 4 tumors arose in the central bronchus and were characterized by a combination of surface exophytic endobronchial papillary proliferation and a submucosal multicystic component with complex architecture. The neoplastic epithelium consisted predominantly of nonciliated stratified columnar cells with ciliated, squamous, and mucinous cells present focally. While 2 tumors (50%) harbored a BRAF V600E mutation by molecular and immunohistochemical analysis, similar to GP/MP, no KRAS, HRAS, AKT1, or PIK3CA mutations were detected in any of the cases. Two patients were treated with limited resection, while 2 patients underwent lobectomy based on the diagnosis of adenocarcinoma or possible squamous cell carcinoma in situ in the preoperative biopsy. All survived without recurrence or metastasis for 23 to 122 months after treatment. SP can develop in the central bronchus as the bronchial counterpart of the salivary gland tumor and should be considered in the differential diagnosis of endobronchial tumors. In addition, some histologic resemblance and frequent BRAF V600E mutation raise the possibility of SP and GP/MP being on the same disease spectrum., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Intraductal carcinoma (IDC) is a salivary gland tumor currently believed to be analogous to breast ductal carcinoma in situ, consisting of a complex neoplastic epithelial proliferation surrounded by a continuous layer of myoepithelial cells presumed to be native and non-neoplastic. Recent molecular insights have shown that there are at least 3 different types of IDC: (1) intercalated duct-like, with frequent NCOA4-RET fusions; (2) apocrine, with multiple mutations similar to salivary duct carcinoma; and (3) mixed intercalated duct-like and apocrine with frequent RET fusions, especially TRIM27-RET. Recent observations (eg, IDC occurring in lymph nodes) have challenged the notion that the myoepithelial cells of IDC are non-neoplastic. Five IDCs with known RET fusions by RNA sequencing were retrieved from the authors' archives, including 4 intercalated duct-like IDCs with NCOA4-RET, and 1 mixed intercalated duct-like/apocrine IDC with TRIM27-RET. A panel of immunohistochemistry antibodies (S100 protein, p63 or p40, mammaglobin, smooth muscle actin, calponin, androgen receptor) was tested. To precisely localize RET split-positive cells, each case was subjected to sequential retrieval of whole-slide imaging data of hematoxylin and eosin (HE) staining, immunofluorescence staining for calponin, and fluorescence in situ hybridization (FISH) for RET. Because NCOA4-RET is an inversion difficult to visualize on conventional RET FISH, a novel 3-color FISH technique was utilized to demonstrate it clearly. In all 5 cases, the proliferative ducts were completely surrounded by a layer of myoepithelial cells that were positive for p63 or p40, smooth muscle actin, and calponin. Using combined HE, calponin immunofluorescence, and RET FISH imaging, the positive signals were unmistakably identified in both calponin-negative ductal cells and peripheral, calponin-positive myoepithelial cells in all 5 cases. Utilizing combined HE, calponin immunofluorescence, and RET FISH imaging, we demonstrated that IDCs with RET fusions harbored this alteration in both the ductal and myoepithelial cells. This is compelling evidence that the myoepithelial cells of IDC are not mere bystanders, but are rather a component of the neoplasm itself, similar to other biphasic salivary gland neoplasms like pleomorphic adenoma and epithelial-myoepithelial carcinoma. This finding raises questions about the appropriate terminology, classification, and staging of IDC., Competing Interests: Conflicts of Interest and Source of Funding: This study was supported in part by the Jane B. and Edwin P. Jenevein, MD Endowment for Pathology at UT Southwestern Medical Center. Supported in part by grants-in-aid for scientific research, MEXT, Japan to H.I. (No. 16H04713). The remaining authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Adenoid cystic carcinoma (AdCC) can demonstrate histologic and immunohistochemical (IHC) overlap with a wide range of salivary and nonsalivary tumors, especially in small biopsy specimens. While MYB fluorescence in situ hybridization (FISH) frequently is used to confirm the diagnosis of AdCC, the pathognomonic MYB-NFIB fusion is only present in 40% to 70% of cases. Likewise, although MYB RNA overexpression is seen in the vast majority of AdCC regardless of translocation status, MYB IHC has shown suboptimal specificity for this diagnosis. In this study, we sought to determine whether a novel chromogenic RNA in situ hybridization (ISH) platform could directly detect MYB RNA overexpression and offer a rapid diagnostic adjunct for AdCC. We performed MYB RNA ISH on 84 cases of AdCC as well as 128 other salivary tumors and 108 basaloid and sinonasal carcinomas that mimic AdCC. MYB RNA ISH was 92% sensitive for AdCC, including 97% of cases with MYB rearrangement and 83% without MYB rearrangement by FISH. It was also 89% specific for AdCC overall, with 95% specificity among other salivary tumors and 81% specificity in basaloid and sinonasal carcinomas. In contrast, MYB IHC was 94% sensitive but just 54% specific for AdCC. Overall, MYB RNA ISH provides superior sensitivity for the diagnosis of AdCC compared with MYB FISH and superior specificity compared with MYB IHC. This assay could provide a useful tool for rapidly confirming the diagnosis of AdCC in formalin-fixed, paraffin-embedded specimens., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Salivary duct carcinoma is a relatively uncommon malignancy of the salivary glands; however, it frequently occurs as a carcinomatous component of carcinoma ex pleomorphic adenoma. We previously reported salivary duct carcinoma with rhabdoid features (SDCRF) as an extremely rare subtype of salivary duct carcinoma, and that it occurred as a salivary counterpart of pleomorphic lobular carcinoma of the breast (PLCB). We collected new cases of SDCRF for this study, in which we examined a total of 17 cases immunohistochemically and genetically. As it is known that PLCB exhibits loss of or aberrant E-cadherin expression and carries nonsense/missense mutations in or deletion of the CDH1 gene, we examined the CDH1 gene status of our SDCRF cases. All of the examined SDCRF cases involved the diffuse proliferation of large ovoid cells with eosinophilic cytoplasm and eccentric nuclei, which displayed reduced cell-cell adhesion. Most cases were positive for pan-cytokeratin, androgen receptor, gross cystic disease fluid protein-15, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1, and WI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 4, whereas they were negative for vimentin. No and decreased/cytoplasmic E-cadherin expression was observed in 11 and 4 of 17 cases, respectively, whereas no and decreased/cytoplasmic β-catenin expression were observed in 10 and 5 of 17 cases, respectively. Among the 11 cases that could be genetically analyzed, a nonsense mutation (1 case), missense mutations (6 cases), and insertions (1 case) were detected in the CDH1 gene. In conclusion, we propose that SDCRF is the salivary counterpart of PLCB due to its morphology and immunophenotype, and the genetic status of CDH1., Competing Interests: Conflicts of Interest and Source of Funding: Supported in part by a Grant-in-Aid for the Medical Research Support Project of Shizuoka Prefectural Hospital Organization in 2019 (to K.K.), as well as by AMED (20ck0106554) (to H.S.) and SRF (to H.S.). The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Oncocytic mucoepidermoid carcinoma (OMEC) is a rare but diagnostically challenging variant of mucoepidermoid carcinoma (MEC). OMEC is notable for differential diagnostic considerations that are raised as a result of overlap with other benign and low-grade oncocytic salivary gland tumors. Diffuse and strong immunoreactivity of p63 protein may be useful in distinguishing OMEC from its mimics. However, focal p63 staining can be present in benign oncytomas. Presence of mucin-containing cells, mucinous cystic formation, and foci of extravasated mucin are considered a hallmark of MEC. True mucocytes may be, however, very few and hardly discernable in OMECs. Recent evidence has shown that most MECs harbor gene fusions involving MAML2. A retrospective review of archived pathology files and the authors' own files was conducted to search for "low-grade/uncertain oncocytic tumor," "oncocytoma," and "oncocytic carcinoma" in the period from 1996 to 2019. The tumors with IHC positivity for p63 and/or p40, and S100 negativity, irrespective of mucicarmine staining, were tested by next-generation sequencing using fusion-detecting panels to detect MAML2 gene rearrangements. Two index cases from consultation practice (A.S. and A.A.) of purely oncocytic low-grade neoplasms without discernible mucinous cells showed a CRTC1-MAML2 fusion using next-generation sequencing, and were reclassified as OMEC. In total, 22 cases of oncocytic tumors, retrieved from the authors' files, and from the Salivary Gland Tumor Registry, harbored the MAML2 gene rearrangements. Presence of mucocytes, the patterns of p63 and SOX10 immunopositivity, and mucicarmine staining were inconsistent findings. Distinguishing OMEC devoid of true mucinous cells from oncocytoma can be very challenging, but it is critical for proper clinical management. Diffuse and strong positivity for p63 and visualization of hidden mucocytes by mucicarmine staining may be misleading and does not always suffice for correct diagnosis. Our experience suggests that ancillary studies for the detection of MAML2 rearrangement may provide useful evidence in difficult cases.

Secretory carcinoma (SC) of the salivary glands is a low-grade carcinoma characterized by a well-defined morphology and immunohistochemical features. ETV6-NTRK3 fusions are detected in the great majority of SCs. Recently, other partners fused to ETV6 have been documented in a small portion of SCs, suggesting the presence of alternative genetic fusion. In this study, we examined the genetic fusion of 9 SCs using fluorescence in situ hybridization, reverse transcription-polymerase chain reaction, and next-generation sequencing (ArcherDx). Classic ETV6 exon 5-NTRK3 exon 15 fusion was detected in 8 of 9 SCs. The remaining tumor was negative for the ETV6-NTRK3 fusion but harbored a novel fusion, CTNNA1 exon 11-ALK in exon 20. Immunohistochemically, pan-TRK was positive in 8 tumors with ETV6-NTRK3 fusion but negative in an ALK-rearranged SC, while ALK was positive only in the ALK-rearranged tumor. Histologically, the ALK-rearranged tumor showed dominant macrocystic architecture. In conclusion, we found a case of SC with CTNNA1-ALK fusion. Because ALK fusion after exon 20 on the ALK side (upstream of the tyrosine kinase domain) has been reported to activate a carcinogenic kinase in various ALK-rearranged tumors, ALK inhibitors may be a possible therapeutic option for ALK-rearranged SC. In addition, ALK immunohistochemistry can be a screening tool for ALK-rearranged SC. This study also expands the molecular spectrum of this tumor beyond the ETV6 gene.

Odontogenic tumors show considerable morphologic heterogeneity and at times the diagnosis can be challenging. Ameloblastoma, the most common odontogenic tumor, can have morphologic similarity to some salivary gland tumors and therefore we sought to identify biomarkers that might aid in the diagnosis by performing transcriptome wide gene expression profiling of 80 odontogenic and salivary gland neoplasms. These data identified the FOXP1/SOX10 expression profile as characteristic of many odontogenic tumors including ameloblastoma but largely absent in salivary gland tumors. We then assessed 173 salivary gland tumors and 108 odontogenic tumors by immunohistochemistry for FOXP1 and SOX10 expression and found that 34/35 (97%) cases of ameloblastomas were diffusely positive for FOXP1 but completely negative for SOX10. None of the basaloid salivary neoplasms (basal cell adenoma, adenoid cystic carcinoma, polymorphous adenocarcinoma, and myoepitheloma) demonstrated FOXP1/SOX10 expression pattern. Taken together, the results of this study suggest that the FOXP1/SOX10 immunophenotype is common in odontogenic tumors including ameloblastoma and might be useful distinguishing these from similar appearing basaloid salivary gland tumors.

Polymorphous adenocarcinoma (PAC) shows histologic diversity with streaming and targetoid features whereas cribriform adenocarcinoma of salivary gland (CASG) demonstrates predominantly cribriform and solid patterns with glomeruloid structures and optically clear nuclei. Opinions diverge on whether CASG represents a separate entity or a variant of PAC. We aimed to assess the level of agreement among 25 expert Head and Neck pathologists in classifying these tumors. Digital slides of 48 cases were reviewed and classified as: PAC, CASG, tumors with ≥50% of papillary architecture (PAP), and tumors with indeterminate features (IND). The consensus diagnoses were correlated with a previously reported molecular alteration. The consensus diagnoses were PAC in 18/48, CASG in16/48, PAP in 3/48, and IND in 11/48. There was a fair interobserver agreement in classifying the tumors (κ=0.370). The full consensus was achieved in 3 (6%) cases, all of which were classified as PAC. A moderate agreement was reached for PAC (κ=0.504) and PAP (κ=0.561), and a fair agreement was reached for CASG (κ=0.390). IND had only slight diagnostic concordance (κ=0.091). PAC predominantly harbored PRKD1 hotspot mutation, whereas CASG was associated with fusion involving PRKD1, PRKD2, or PRKD3. However, such molecular events were not exclusive as 7% of PAC had fusion and 13% of CASG had mutation. In conclusion, a fair to moderate interobserver agreement can be achieved in classifying PAC and CASG. However, a subset (23%) showed indeterminate features and was difficult to place along the morphologic spectrum of PAC/CASG among expert pathologists. This may explain the controversy in classifying these tumors.

Salivary duct carcinoma (SDC) is a rare, aggressive malignancy that histologically resembles high-grade mammary duct carcinoma. Because of the rarity of this entity, data verifying the association between histologic features and patient survival are limited. We conducted a comprehensive histologic review of 151 SDC cases and performed an analysis of the association between various histomorphologic parameters and the clinical outcome with the aim of developing a histologic risk stratification model that predicts the prognosis of SDC patients. A multivariate analysis revealed that prominent nuclear pleomorphism (overall survival [OS]: P=0.013; progression-free survival [PFS]: P=0.019), ≥30 mitoses/10 HPF (PFS: P=0.013), high tumor budding (OS: P=0.011; PFS: P<0.001), and high poorly differentiated clusters (OS: P<0.001; PFS: P<0.001) were independent prognostic factors. Patients with vascular invasion demonstrated a marginally significant association with shorter PFS (P=0.064) in a multivariate analysis. We proposed a 3-tier histologic risk stratification model based on the total number of positive factors among 4 prognostically relevant parameters (prominent nuclear pleomorphism, ≥30 mitoses/10 HPF, vascular invasion, and high poorly differentiated clusters). The OS and PFS of patients with low-risk (0 to 1 point) (23% of cases), intermediate-risk (2 to 3 points) (54% of cases), and high-risk (4 points) (23% of cases) tumors progressively deteriorated in this order (hazard ratio, 2.13 and 2.28, and 4.99 and 4.50, respectively; Ptrend<0.001). Our histologic risk stratification model could effectively predict patient survival and may be a useful aid to guide clinical decision-making in relation to the management of patients with SDC.

Mammary analogue secretory carcinoma (MASC) is a relatively recently described salivary gland adenocarcinoma characterized by ETV6-NTRK3 gene fusion and in most cases indolent clinical behavior. The majority of tumors show an admixture of microcystic, solid, and tubular growth patterns but only a few cases with dominant macrocystic growth have been reported. We report 15 cases of macrocystic MASC. There were 11 men and 4 women (17 to 88 y age range, average 47 y). The patients presented with a painless cystic mass, the majority in the region of the parotid gland (n=13), as well as in submandibular gland (n=1) and the neck (n=1). All tumors were circumscribed measuring 1.0 to 4.0 cm in greatest diameter (mean: 1.75 cm). Twelve tumors were unilocular, while 3 were multilocular. The cystic spaces were predominantly lined by a single epithelial cell layer with focal areas in which the epithelium was multilayered with papillary and hobnail features. In 3 of the cases there were more solid foci of intracystic tumor characterized by papillary and/or microcystic growth. The neoplastic cells were round to oval with hyperchromatic to vesicular nuclei with centrally located nucleoli and eosinophilic or vacuolated cytoplasm. Tumor cells showed strong positivity for S100 protein and mammaglobin, while DOG1 was uniformly negative. A minority of cases showed focal p63 reactivity predominantly limited to the periphery of the cystic lining. ETV6 gene rearrangement was identified in 9 cases. Macrocystic MASC can simulate benign and malignant salivary gland lesions and needs to be included in the differential diagnosis of cystic lesions in the head and neck. To the best of our knowledge, our report represents the first series of macrocystic MASCs wholly focusing on this unusual variant.

Intraductal carcinoma (IC) is the new WHO designation for tumors previously encompassed by "low-grade cribriform cystadenocarcinoma" and "low-grade salivary duct carcinoma." The relationship of IC to salivary duct carcinoma (SDC) is controversial, even though they are considered to be distinct entities. IC is a rare low-grade malignant salivary gland neoplasm with histopathological features reminiscent of atypical ductal hyperplasia or ductal carcinoma in situ of the breast, showing diffuse S100 protein and mammaglobin positivity, while it is partially defined genetically. Recently, RET rearrangements including NCOA4-RET and TRIM27-RET have been described in IC. Here, we genetically characterize the largest cohort of IC to date (33 cases) including 8 cases with focal or widespread invasive growth and 1 case with lymph node metastasis. Thirty-three cases of IC were analyzed by next-generation sequencing (NGS) using the FusionPlex Solid Tumor kit (ArcherDX). Identified gene fusions were confirmed using fluorescence in situ hybridization break-apart and fusion probes and an reverse transcription polymerase chain reaction designed specifically for the detected breakpoints. Ten cases of SDC were analyzed for comparison using NGS panels that detect mutations and fusion transcripts. NGS analysis detected an NCOA4-RET fusion transcript in 11 cases of intercalated duct-type IC joining exon 7 or 8 of NCOA4 gene and exon 12 of the RET gene. Eight cases of IC had an invasive growth pattern, including one with widespread invasion and lymph node metastasis. Three invasive ICs harbored an NCOA4-RET fusion transcript, while 1 case was negative, and 2 cases were not analyzable. In addition, a novel TRIM27-RET fusion transcript between exon 3 of TRIM27 and exon 12 of RET was identified in 2 cases of IC with apocrine features, and one of them displayed invasive growth. Two IC cases with invasive growth harbored novel fusions TUT1-ETV5 and KIAA1217-RET, respectively. A total of 42.4% of the cases in this series of IC harbored fusions involving RET. Such fusion transcripts were not detected in any of the 10 SDC cases. We have confirmed NCOA4-RET as a predominant fusion in intercalated duct-type IC, including 3 cases with invasive growth pattern. A novel finding in our series was a case of widely invasive intercalated duct-type IC, with a single lymph node metastasis that revealed an NCOA4-RET fusion transcript. We also demonstrated that a subset of apocrine ICs harbored a TRIM27-RET gene fusion, including one case with invasive growth. In contrast, neither NCOA4-RET nor TRIM27-RET fusions were detected in any tested SDCs. Thus, the distinct molecular findings in IC and SDC support that the tumors are separate malignant salivary tumor entities. The presence of tumor-type-specific NCOA4-RET or TRIM27-RET translocations in a subset of widely invasive carcinomas with intercalated duct-like immunoprofiles suggests that a recharacterization of IC including its redesignation as "intercalated duct carcinoma, invasive or noninvasive" may be appropriate.

Pleomorphic adenoma (PA) is the most common salivary gland neoplasm. On a molecular level PA is characterized by a translocation involving PLAG1 or HMGA2. PA is considered to be a benign tumor although it can undergo malignant transformation. Alternatively, cases of histologically benign PA "metastasizing" to lymph nodes or distant body sites are well documented. Several theories have been proposed to explain this behavior. However, there is a lack of molecular data available to assess the relationship of metastasizing PA (MPA) and their benign counterparts. In this study we describe 4 cases of MPAs and perform the first molecular study linking them to conventional PA. The index case was identified in the course of routine clinical practice, while the other cases were retrieved from the archives of the authors. Slides were reviewed to confirm the diagnosis of both the primary/recurrent tumor and the metastasis. Fluorescence in situ hybridization (FISH) was performed in all cases and RNA sequencing was performed on the index case. In all cases there was a history of recurrent PA involving the parotid. Lymph node metastases were identified in 2 cases; non-lymph node metastases were identified in 3 cases. All the metastases were histologically benign. RNA sequencing performed on the index case demonstrated a novel HMGA2-TMTC2 translocation, which was confirmed by separate FISH break-apart assays for both genes. FISH performed on the remaining cases demonstrated rearrangement of PLAG1 in all 3 cases. This study demonstrates that MPA harbors the same disease-defining molecular hallmark as their benign counterparts.

Salivary gland adenocarcinoma not otherwise specified (NOS) is a heterogenous group, likely containing distinct tumors not yet characterized. A growing number of low to intermediate-grade salivary carcinomas are now known to harbor tumor-specific gene fusions. On occasion, identifying a novel fusion allows for recognition of a new salivary tumor type, in addition to representing a potential diagnostic tool. We sought to characterize a distinctive salivary gland adenocarcinoma that would previously have been regarded as adenocarcinoma NOS. On the basis of the recognition of 5 morphologically identical, distinct low-grade salivary adenocarcinomas, we used targeted RNA sequencing (RNA-Seq) to determine whether these could be differentiated from other fusion-associated salivary gland tumors. RNA-Seq was performed on all 5 low-intermediate grade adenocarcinomas NOS with near-identical histologic appearances, as well as 23 low-intermediate grade control adenocarcinoma NOS cases that did not resemble the index cases. All 5 index cases harbored a novel MEF2C-SS18 gene fusion, which was independently confirmed by reverse transcriptase-polymerase chain reaction. The MEF2C-SS18-positive cases arose in the oral cavity (4/5) and parotid gland (1/5) of 3 women and 2 men ranging from 21 to 80 years (mean: 46) and shared near-identical histologic features: intercalated duct-like cells with eosinophilic to clear cytoplasm and small, uniform oval nuclei, infiltrative microcysts and cords, abundant intraluminal secretions, and cellular fibromyxoid stroma. Mitotic rates were low; necrosis was absent. All MEF2C-SS18-positive tumors were positive for S100 and p63 and negative for p40, smooth muscle actin, calponin, and mammaglobin. One of the 23 control cases, a parotid tumor, was found to contain a SS18-ZBTB7A gene fusion; it demonstrated similar, but not identical histologic and immunophenotypic features compared with the MEF2C-SS18 cases. The remaining control cases were negative for SS18 and MEF2C rearrangements. A novel MEF2C-SS18 gene fusion and unique histologic and immunophenotypic features characterize a heretofore undefined low-grade salivary adenocarcinoma for which we propose the term "microsecretory adenocarcinoma." RNA-Seq helped establish this entity as a distinct tumor type, and identified one possibly related case with a different SS18-related fusion. The recognition of microsecretory adenocarcinoma and its separation from other adenocarcinomas NOS will facilitate a more complete understanding of the clinical and pathologic characteristics of this previously unrecognized neoplasm.

Adenomyoepitheliomas (AME) of the breast and epithelial-myoepithelial carcinomas (EMCs) of salivary gland are morphologically similar tumors defined by the presence of a biphasic population of ductal epithelial elements mixed with myoepithelial cells. We sought to explore the molecular profile of AMEs and determine whether they might also share the PLAG1, HMGA2, and HRAS alterations seen in EMCs. Tumor tissue from 19 AMEs was sequenced and analyzed using Ion AmpliSeq Cancer Hotspot Panel v2 covering ∼2800 COSMIC mutations across 50 cancer-related genes. Cases were additionally screened by FISH for PLAG1 and HMGA2 rearrangements. Of 19 AMEs (12 benign; 7 malignant), 2 cases failed the DNA extraction. Of the remaining 17 cases, 14 had at least one nonsynonymous mutation identified. The most common mutations were in PIK3CA (6/17) and AKT1 (5/17), which were mutually exclusive. Two tumors demonstrated mutations in APC, while 1 demonstrated an STK11 mutation. Mutations in ATM, EGFR, FGFR3 or GNAS were identified in 4 cases with concurrent AKT1 mutations. HRAS mutation co-occurring with PIK3CA mutation was noted in 1 case of ER-negative malignant AME. While 2 cases harbored alterations in HMGA2, none was positive for PLAG1 rearrangement. Our findings confirm that breast AMEs are genetically heterogeneous exhibiting recurrent mutually exclusive mutations of PIK3CA and AKT1 in a majority of cases. HRAS mutations co-occur with PIK3CA mutations in ER-negative AMEs and may possibly be linked to clinically aggressive behavior. We identified hotspot mutations in additional genes (APC, STK11, ATM, EGFR, FGFR3, and GNAS). We report the presence of HMGA2 alterations in 2/16 AMEs, supporting their relationship with EMC of salivary glands in at least a subset of cases. PIK3CA, AKT1 and HRAS may serve as potential actionable therapeutic targets in clinically aggressive AMEs.

Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy, but categorization is complicated by variability in grading systems and uncertain prognostic significance of MAML2 rearrangement. The aims of this study were to determine the prognostic significance of MEC grading systems and MAML2 rearrangement status. Fifty-three carcinomas originally diagnosed as MEC (45 primary; 8 recurrent) of major and minor salivary glands were graded according to modified Healey, Brandwein, AFIP, and Katabi systems. Fluorescence in situ hybridization for MAML2 rearrangement was performed. Clinical features and outcomes were recorded. Twenty-five (47%) carcinomas scored the same in all grading systems. The most common histologic feature leading to a diagnosis of intermediate grade was isolated solid growth. Brandwein assigned the highest percentage of high grade (29%) and AFIP the highest percentage of low grade (80%). MAML2 was rearranged in 37/46 (80%) cases. Forty-three (81%) were morphologically compatible with MEC, and these were more likely to be low-intermediate grade and MAML2-rearranged. Of primary carcinomas, 6 (13%) recurred. Statistically significant univariate risk factors for recurrence included non-MEC morphology, stage T4, and high Brandwein grade. Margin status, MAML2 rearrangement, and isolated solid growth were not predictive of recurrence. A binary grading system (Brandwein high vs. low-plus-intermediate) could be considered to better reflect biological behavior in MEC. Our study confirms that MAML2 wildtype tumors more likely represent high grade non-MECs, and prior studies demonstrating worse prognosis in MAML2-nonrearranged MECs may be diluted by high-grade non-MECs.

Epithelial-myoepithelial carcinoma (EMC) is a rare salivary gland tumor that is histologically characterized by biphasic tubular structures composed of inner ductal and outer clear myoepithelial cells. Because of its histologic variety, it is sometimes challenging to make an accurate diagnosis, and useful ancillary tests are essential for this purpose. We investigated 87 cases of EMC arising in the major and minor salivary glands and seromucinous glands in the nasal cavity or bronchus to describe the histologic features and mutation status of selected key oncogenes. Classic EMC accounted for 40.2% of all cases. Other cases showed various growth patterns and cytologic features in addition to the typical histology; cribriform patterns, a basaloid appearance, and sebaceous differentiation were relatively common (17.2% to 18.4%), whereas oncocytic/apocrine, papillary-cystic, double-clear, squamous, psammomatous, Verocay-like, and high-grade transformation were rare. HRAS mutations were found in 82.7% of EMCs and were concentrated in codon 61. There was no significant correlation between the HRAS mutation status and the histology. No EMC ex pleomorphic adenoma cases had HRAS mutations. PIK3CA and/or AKT1 mutations were the second most frequent mutations (20.7%, 6.5%, respectively) and almost always cooccurred with HRAS mutations. It is noteworthy that the HRAS mutation was not identified in any salivary gland tumor entities manifesting EMC-like features, including adenoid cystic carcinoma, pleomorphic adenoma, basal cell adenoma/adenocarcinoma, and myoepithelial carcinoma. We conclude that HRAS mutations are a frequent tumorigenic gene alteration in EMC, despite its histologic diversity. This study provides further insight into strategies for diagnosing EMC and discriminating it from its mimics.

Myoepithelial carcinoma (MECA) is an underrecognized challenging entity with a broad morphologic spectrum. Misinterpreting MECA is not uncommon as distinguishing it from its mimics, especially cellular myoepithelial-rich pleomorphic adenoma (PA), can be difficult. We described 21 histologically challenging cases of MECAs (16 MECA ex-PA and 5 MECA de novo). All MECAs ex-PA were intracapsular or minimally invasive except for 3 cases. Eighteen (86%) were initially misinterpreted as benign neoplasms, including PA (10), atypical PA (5), and myoepithelioma (3). The remaining 3 were initially diagnosed as malignant (MECA ex-PA) but were histologically challenging. Histologic features that were found most helpful in recognizing the malignant nature of MECA included: uniformly cellular myoepithelial proliferation with an expansile nodular lobulated pattern (all cases) and alternate hypocellular and hypercellular zonal distribution (76% of cases). Among the 16 MECA patients with follow-up, 14 (87.5%) progressed: 10 developed local recurrence and 5 distant metastases. In contrast, only one of 33 patients with cellular PA (control group) recurred locally. Ten of the 14 MECAs that progressed were MECA ex-PA, and 12 (85%) had an initial benign diagnosis. Two patients with MECA ex-PA died of their disease; one had an initial diagnosis of PA. MECA is a histologically challenging entity that closely mimics PA and seems to carry a significant risk of recurrence. Areas of clonal appearing cellular myoepithelial growth with an expansile nodular lobulated pattern and zonal cellular distribution distinguish the majority of MECAs and may serve as useful diagnostic histologic features to differentiate MECA from its benign mimics.

The spectrum of tumors arising in the salivary glands is wide and has recently been shown to harbor a network of tumor-specific fusion genes. Acinic cell carcinoma (AciCC) is one of the more frequently encountered types of salivary gland carcinoma, but it has remained a genetic orphan until recently when a fusion between the HTN3 and MSANTD3 genes was described in one case. Neither of these 2 genes is known to be implicated in any other malignancy. This study was undertaken to investigate whether the HTN3-MSANTD3 fusion is a recurrent genetic event in AciCC and whether it is a characteristic of one of its histological variants. Of the 273 AciCCs screened, 9 cases showed rearrangement of MSANTD3 by break-apart fluorescence in situ hybridization, 2 had 1 to 2 extra signals, and 1 had gain, giving a total of 4.4% with MSANTD3 aberrations. In 6 of 7 available cases with MSANTD3 rearrangement, the HTN3-MSANTD3 fusion transcript was demonstrated with real-time polymerase chain reaction. Histologically, all fusion-positive cases were predominantly composed of serous tumor cells growing in solid sheets, with serous tumor cells expressing DOG-1 and the intercalated duct-like cell component being CK7 positive and S-100 positive in 6/9 cases. All but one case arose in the parotid gland, and none of the patients experienced a recurrence during follow-up. In contrast, the case with MSANTD3 gain metastasized to the cervical lymph nodes and lungs. In conclusion, we find the HTN3-MSANTD3 gene fusion to be a recurrent event in AciCC with prominent serous differentiation and an indolent clinical course.

Adamantinoma-like Ewing sarcoma (ALES) is a rare tumor that demonstrates the EWSR1-FLI1 translocation characteristic of Ewing sarcoma despite overt epithelial differentiation including diffuse expression of cytokeratins and p40. Most cases of ALES described to date have occurred in the head and neck where they can mimic a wide range of small round blue cell tumors. Because distinguishing ALES from basaloid salivary gland carcinomas can be particularly difficult, we analyzed a series of 10 ALESs that occurred in the salivary glands with the aim of identifying features that allow for better recognition of this entity. The salivary ALESs included 8 parotid gland and 2 submandibular gland tumors in patients ranging from 32 to 77 years (mean: 52 y). Nine were initially misclassified as various epithelial neoplasms. Although these tumors displayed the basaloid cytology, rosette formation, infiltrative growth, and nuclear monotony characteristic of ALES, peripheral palisading and overt keratinization were relatively rare in this site. Salivary ALESs not only displayed positivity for AE1/AE3, p40, and CD99, but also demonstrated a higher proportion of synaptophysin reactivity than has been reported for nonsalivary ALESs. These morphologic and immunohistochemical findings make ALES susceptible to misclassification as various other tumors including basal cell adenocarcinoma, adenoid cystic carcinoma, squamous cell carcinoma, NUT carcinoma, large cell neuroendocrine carcinoma and myoepithelial carcinoma. Nevertheless, monotonous cytology despite highly infiltrative growth and concomitant positivity for p40 and synaptophysin can provide important clues for consideration of ALES, and identification of the defining EWSR1-FLI1 translocations can confirm the diagnosis.

"Mucoepidermoid carcinoma (MEC)" has been accepted as a synonym for pancreatic adenosquamous carcinoma (ASC). Pancreatic ASC can show salivary gland-type MEC-like morphology. CRTC1/3-MAML2 fusion gene is a characteristic molecular feature of MEC of the salivary gland. We conducted this study to clarify whether the pancreatic ASC with salivary gland-type MEC-like morphology (Pan-MEC) is a pancreatic counterpart of salivary gland-type MEC (Sal-MEC). We retrospectively analyzed 37 pancreatic ASCs including 16 Pan-MECs and 21 tumors without MEC-like features (ASC-NOS [not otherwise specified]), and we investigated (1) clinicopathologic features, (2) the presence of CRTC1/3-MAML2 fusion gene by reverse transcription polymerase chain reaction, (3) the presence of rearrangement of MAML2 gene by fluorescence in situ hybridization, and (4) mucin core proteins by immunohistochemistry. We also compared 16 Pan-MECs with 20 Sal-MECs by immunohistochemistry for mucin core protein. There were no significant differences of any clinicopathologic characteristics and survival analysis between the Pan-MECs and ASCs-NOS. Of note, the pancreatic ASCs (including Pan-MEC and ASC-NOS) were significantly more aggressive than conventional pancreatic ductal adenocarcinoma. In addition, all Pan-MECs were histologically high-grade. CRTC1/3-MAML2 fusion gene and MAML2 gene rearrangement were not detected in any ASCs including Pan-MECs. There were significant differences of MUC5AC and MUC6 between the Pan-MECs and Sal-MECs, but no significant differences of mucin core protein between the Pan-MECs and pancreatic ASCs-NOS. Pan-MEC is histologically and biologically high-grade and unrelated to CRTC1/3-MAML2 fusion gene, unlike Sal-MEC which is related to CRTC1/3-MAML2 fusion gene. Pan-MEC is not a pancreatic counterpart of CRTC1/3-MAML2 fusion gene-related Sal-MEC.

Intraductal carcinoma (IC) is the new World Health Organization designation for tumors previously called "low-grade cribriform cystadenocarcinoma" and "low-grade salivary duct carcinoma." The relationship of IC to salivary duct carcinoma is controversial, but they now are considered to be distinct entities. IC is a rare low-grade malignant salivary gland neoplasm with features similar to mammary atypical ductal hyperplasia or ductal carcinoma in situ, that shows diffuse S100 protein and mammaglobin positivity and is only partially defined genetically. (Mammary analogue) secretory carcinoma harboring ETV6-NTRK3, and in rare cases ETV6-RET fusion, shares histomorphologic and immunophenotypical features with IC. Recently, RET rearrangements and NCOA4-RET have been described in IC, suggesting a partial genetic overlap with mammary analogue secretory carcinoma. Here, we genetically characterize the largest cohort of IC to date to further explore this relationship. Seventeen cases of IC were analyzed by next-generation sequencing using the FusionPlex Solid Tumor kit (ArcherDX). Identified fusions were confirmed using fluorescence in situ hybridization break apart and, in some cases, fusion probes, and a reverse transcription polymerase chain reaction designed specifically to the detected breakpoints. All analyzed cases were known to be negative for ETV6 rearrangement by fluorescence in situ hybridization and for ETV6-NTRK3 fusion by reverse transcription polymerase chain reaction. Next-generation sequencing analysis detected a NCOA4-RET fusion transcript joining exon 7 or 8 of NCOA4 gene and exon 12 of RET gene in 6 cases of intercalated duct type IC; and a novel TRIM27-RET fusion transcript between exons 3 and 12 in 2 cases of salivary gland tumors displaying histologic and immunohistochemical features typical of apocrine IC. A total of 47% of IC harbored a fusion involving RET. In conclusion, we have confirmed the presence of NCOA4-RET as the dominant fusion in intercalated duct type IC. A novel finding in our study has been a discovery of a subset of IC patients with apocrine variant IC harboring a novel TRIM27-RET.

The spectrum of low-grade intraductal papillary proliferations of the salivary glands is heterogenous, and reproducible morphologic diagnostic criteria have not yet been established. Recognized types are sialadenoma papilliferum, inverted ductal papilloma, and intraductal papilloma, but some lesions have been possibly included in the morphologic spectrum of cystadenoma or low-grade intraductal carcinomas. We herein present detailed morphologic, immunophenotypic, and genotypic features of 3 minor salivary gland neoplasms affecting 2 men (aged 65 and 71 y) and 1 woman (aged 78 y). They ranged in size from 1 to 2.5 cm. All tumors showed atypical papillary intraductal growth that presented either as uninodular/unicystic lesions (intraductal papilloma-like; n=2) or as a discontinuous growth along the ductal system in a manner similar to pancreatic intraductal papillary mucinous neoplasm (n=1). Variable cytologic and architectural atypia was observed, ranging from bland intraductal papilloma-like features, to areas mimicking atypical ductal hyperplasia and low-grade ductal carcinoma in situ of the breast. Amplicon-based massive parallel sequencing revealed an identical AKT1 p.Glu17Lys mutation in all 3 cases, but absence of concurring mutations in other genes of the RAS or PI3K pathway. This small series represents the first genetic study on salivary intraductal papillary neoplasms. Our cases showed significant variation in the degree of cytologic and architectural atypia, which overlaps with intraductal papillomas at the one end and with low-grade intraductal carcinoma at the other end of the spectrum, suggesting a disease continuum. As the full biological and morphologic characteristics of these ductal papillary lesions remain to be defined, the noncommitted term "intraductal papillary neoplasms" might be more appropriate. Our novel genetic findings mirror similar activating mutations of AKT1 and other PI3K pathway members in intraductal papillary lesions of the breast and anogenital glands.

Secretory carcinoma of the salivary glands, also known as mammary analogue secretory carcinoma, is a recently described tumor characterized by generally indolent clinical behavior and recurrent ETV6-NTRK3 fusions. However, a small subset of recent cases with high-grade histology, aggressive behavior, or alternate molecular findings are expanding the spectrum of this entity. In this case, a 59-year-old female presented with an infiltrative submandibular gland tumor that was originally classified as a high-grade acinic cell carcinoma, papillary-cystic variant. She developed persistent local disease and, 11 years after initial presentation, was found to have widespread metastases. Rereview of her primary tumor highlighted microcystic, papillary, and solid architecture, eosinophilic cytoplasm, vesicular nuclei with prominent nucleoli, abundant mitotic figures, and necrosis. Immunostains showed the tumor cells to be positive for S100 and mammaglobin and negative for DOG-1, and fluorescence in situ hybridization highlighted an ETV6 rearrangement, supporting a diagnosis of high-grade secretory carcinoma. Finally, next-generation sequencing demonstrated a novel ETV6-MET fusion. To our knowledge, this is the first ETV6-MET fusion reported in secretory carcinoma. This finding further expands the definition of secretory carcinoma while carrying implications for selecting appropriate targeted therapy.

Intraductal carcinoma (IC) is the World Health Organization designation for lesions previously called low-grade cribriform cystadenocarcinoma. The relationship of IC to salivary duct carcinoma (SDC) is controversial, but currently these are considered distinct entities. It is hypothesized that IC and SDC should have different genomic signatures that may be identifiable by next-generation sequencing. A total of 23 ICs were identified: 14 pure IC and 9 invasive carcinomas with an intraductal component. Five invasive carcinomas were subjected to next-generation paired-end RNA sequencing. Data analysis was performed using FusionSeq and Mutation detection algorithms (MuTect and VarScan) for variant callers. Gene fusion candidates were validated by fluorescence in situ hybridization and reverse transcription polymerase chain reaction, and mutations by Sanger sequencing. Among the 9 invasive carcinomas, all except 1 were apocrine SDCs with an intraductal component. The remaining case showed typical intercalated duct type IC with invasive adenocarcinoma. The 14 pure ICs had typical intercalated duct features (2 showed hybrid intercalated/apocrine features). RNA sequencing predicted a NCOA4-RET fusion, confirmed by reverse transcription polymerase chain reaction, in the intercalated duct type IC invasive component. Six additional cases of pure IC showed RET rearrangement by fluorescence in situ hybridization (7/15=47%). No apocrine carcinomas showed RET rearrangement. RNA sequencing and Sanger sequencing identified PIK3CA (p.E545K/p.H1047R) and/or HRAS (p.Q61R) hotspot mutations in 6 of 8 (75%) apocrine carcinomas. In conclusion, 2 distinctive types of intraductal lesions are emerging based on molecular analysis. Classic intercalated type ICs commonly harbor fusions involving RET and rarely show widespread invasion. Apocrine intraductal lesions are typically associated with widespread invasion with no pure examples and show similar PIK3CA and HRAS mutations to SDC.

Epithelial-myoepithelial carcinoma (EMC) is a malignant salivary gland neoplasm comprised of a biphasic arrangement of inner luminal ductal cells and outer myoepithelial cells. Adenoid cystic carcinoma (AdCC) is also a biphasic tumor comprised of ductal and myoepithelial cells, but these components tend to be arranged in a more cribriform pattern. The occurrence of "hybrid carcinomas" that show mixed patterns of EMC and AdCC raises questions about the relationship of these morphologically overlapping but clinically distinct tumors. AdCCs frequently harbor MYB-NFIB gene fusions. Mapping of EMCs (including hybrid forms with an AdCC component) for this fusion could help clarify the true nature of EMC as a distinct entity or simply as some variant form of AdCC. Twenty-nine cases of EMC were evaluated including 15 classic low-grade EMCs, 7 intermediate-grade EMCs, 2 EMCs with myoepithelial anaplasia, 1 EMC with high-grade transformation, and 4 hybrid EMCs with an AdCC component. Break apart fluorescence in situ hybridization for MYB was performed, as was MYB immunohistochemistry. For the hybrid carcinomas and those with high-grade transformation, the divergent tumor components were separately analyzed. A MYB translocation was identified in 5 of 28 (18%) tumors including 3 of 4 (75%) hybrid carcinomas and 2 of 7 (29%) intermediate-grade EMCs. For the positive hybrid carcinomas, the fusion was detected in both the EMC and AdCC components. The MYB fusion was not detected in any of the classic EMCs (0/15) or in any of the EMCs with myoepithelial anaplasia (0/2) or high-grade transformation (0/1). The fluorescence in situ hybridization assay was unsuccessful in 1 case. MYB immunostaining was seen in 5 of 5 fusion-positive cases, and also 9 of 23 fusion-negative tumors. Classic low-grade EMCs are genetically distinct from AdCCs in that they do not harbor MYB fusions. The presence of a MYB fusion in EMCs showing hybrid features of AdCC or exhibiting highly infiltrative growth points to a subset of these tumors that may well be true AdCCs masquerading as EMCs.

Clear cell carcinoma (CCC) is a low-grade malignancy that commonly arises in minor salivary glands of the oropharynx and other sites. EWSR1-ATF1 gene fusions seem to be specific for this salivary neoplasm. Testing for EWSR1-ATF1 has expanded the histologic spectrum of CCC. As one important example, many CCCs have a predominantly squamous phenotype with few clear cells, a finding that can cause confusion with squamous cell carcinoma (SqCC). P16 immunohistochemical staining to determine human papillomavirus (HPV) status has become standard practice for all oropharyngeal carcinomas showing squamous differentiation. The purpose of this study was to determine whether this practice could contribute to the difficulty in distinguishing CCC from p16-positive SqCC. The authors' surgical pathology archives were searched for cases of CCC. All cases were evaluated with p16 immunohistochemistry, high-risk HPV RNA in situ hybridization (ISH), and EWSR1 gene break-apart fluorescence ISH. Sixteen CCCs were identified. All harbored an EWSR1 rearrangement. Eleven patients were women and 5 were men. They ranged in age from 30 to 85 years (mean, 58 y). The CCCs arose in the oropharynx (tongue base or tonsil) (n=8, 50%), oral cavity (n=4, 25%), and nasopharynx (n=4, 25%). Each case demonstrated clear cells, but the proportion was highly variable (10% to 90%, mean 48%), with 7 of 16 cases having <50% clear cells. Submitted diagnoses included SqCC (n=3) and mucoepidermoid carcinoma (n=2). Of the 3 patients diagnosed with SqCC, 1 was scheduled to undergo chemoradiation, and 1 had already completed chemoradiation. All 16 CCCs demonstrated p16 staining, with the percentage of p16-positive cells ranging from ≥70% (n=2), 50% to 69% (n=3), and 10% to 49% (n=11). Staining was cytoplasmic and nuclear. All cases were negative for high-risk HPV by RNA ISH. CCCs regularly show squamous features, often lack prominent clear cell changes, frequently arise in the oropharynx, and invariably show p16 staining. These features may cause confusion with SqCC, particularly HPV-related oropharyngeal SqCC. P16 staining is not to be taken as unequivocal evidence of an HPV-related SqCC, even for carcinomas showing squamous differentiation and originating in the oropharynx. Failure to recognize this pitfall could result in overly aggressive treatment of a low-grade carcinoma.

Salivary gland neoplasms are a morphologically heterogenous group of lesions that are often diagnostically challenging. In recent years, considerable progress in salivary gland taxonomy has been reached by the discovery of tumor type-specific fusion oncogenes generated by chromosome translocations. This review describes the clinicopathologic features of a selected group of salivary gland carcinomas with a focus on their distinctive genomic characteristics. Mammary analog secretory carcinoma is a recently described entity characterized by a t(12;15)(p13;q25) translocation resulting in an ETV6-NTRK3 fusion. Hyalinizing clear cell carcinoma is a low-grade tumor with infrequent nodal and distant metastasis, recently shown to harbor an EWSR1-ATF1 gene fusion. The CRTC1-MAML2 fusion gene resulting from a t(11;19)(q21;p13) translocation, is now known to be a feature of both low-grade and high-grade mucoepidermoid carcinomas associated with improved survival. A t(6;9)(q22-23;p23-34) translocation resulting in a MYB-NFIB gene fusion has been identified in the majority of adenoid cystic carcinomas. Polymorphous (low-grade) adenocarcinoma and cribriform adenocarcinoma of (minor) salivary gland origin are related entities with partly differing clinicopathologic and genomic profiles; they are the subject of an ongoing taxonomic debate. Polymorphous (low-grade) adenocarcinomas are characterized by hot spot point E710D mutations in the PRKD1 gene, whereas cribriform adenocarcinoma of (minor) salivary glands origin are characterized by translocations involving the PRKD1-3 genes. Salivary duct carcinoma (SDC) is a high-grade adenocarcinoma with morphologic and molecular features akin to invasive ductal carcinoma of the breast, including HER2 gene amplification, mutations of TP53, PIK3CA, and HRAS and loss or mutation of PTEN. Notably, a recurrent NCOA4-RET fusion has also been found in SDC. A subset of SDC with apocrine morphology is associated with overexpression of androgen receptors. As these genetic aberrations are recurrent they serve as powerful diagnostic tools in salivary gland tumor diagnosis, and therefore also in refinement of salivary gland cancer classification. Moreover, they are promising as prognostic biomarkers and targets of therapy.

ETV6 gene abnormalities are well described in tumor pathology. Many fusion partners of ETV6 have been reported in a variety of epithelial, mesenchymal, and hematological malignancies. In salivary gland tumor pathology, however, the ETV6-NTRK3 translocation is specific for (mammary analog) secretory carcinoma, and has not been documented in any other salivary tumor type. The present study comprised a clinical, histologic, and molecular analysis of 10 cases of secretory carcinoma, with typical morphology and immunoprofile harboring a novel ETV6-RET translocation.

We hypothesized that there is a relationship between the preexisting pleomorphic adenoma [PA]), histologic grade of epithelial-myoepithelial carcinomas (EMCAs), and genetic alterations. EMCAs (n=39) were analyzed for morphologic and molecular evidence of preexisting PA (PLAG1, HMGA2 status by fluorescence in situ hybridization, FISH, and FGFR1-PLAG1 fusion by next-generation sequencing, NGS). Twenty-three EMCAs were further analyzed by NGS for mutations and copy number variation in 50 cancer-related genes. On the basis of combined morphologic and molecular evidence of PA, the following subsets of EMCA emerged: (a) EMCAs with morphologic evidence of preexisting PA, but intact PLAG1 and HMGA2 (12/39, 31%), (b) Carcinomas with PLAG1 alterations (9/39, 23%), or (c) HMGA2 alterations (10/39, 26%), and (d) de novo carcinomas, without morphologic or molecular evidence of PA (8/39, 21%). Twelve high-grade EMCAs (12/39, 31%) occurred across all subsets. The median disease-free survival was 80 months (95% confidence interval, 77-84 mo). Disease-free survival and other clinicopathologic parameters did not differ by the above defined subsets. HRAS mutations were more common in EMCAs with intact PLAG1 and HMGA2 (7/9 vs. 1/14, P<0.001). Other genetic abnormalities (TP53 [n=2], FBXW7 [n=1], SMARCB1 deletion [n=1]) were seen only in high-grade EMCAs with intact PLAG1 and HMGA2. We conclude that most EMCAs arose ex PA (31/39, 80%) and the genetic profile of EMCA varies with the absence or presence of preexisting PA and its cytogenetic signature. Progression to higher grade EMCA with intact PLAG1 and HMGA2 correlates with the presence of TP53, FBXW7 mutations, or SMARCB1 deletion.

Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy. Recent studies have shown that most MECs harbor gene fusions involving MAML2-an alteration that appears to be specific for MEC, a finding that could be diagnostically useful. While most cases of MEC are histologically straightforward, uncommon variants can cause considerable diagnostic difficulty. We present 2 variants of MEC for which MAML2 studies were crucial in establishing a diagnosis: a previously undescribed ciliated variant, and the recently described Warthin-like variant. All cases of ciliated and Warthin-like MEC were retrieved from the archives of The Johns Hopkins Hospital. Break-apart fluorescence in situ hybridization for MAML2 was performed on all cases. One ciliated MEC and 6 Warthin-like MECs were identified. The ciliated MEC presented as a 4.6 cm cystic lymph node metastasis originating from the tongue base in a 47-year-old woman. The Warthin-like MECs presented as parotid masses ranging in size from 1.2 to 3.3 (mean, 2.7 cm) in 4 women and 2 men. The ciliated MEC consisted of macrocystic spaces punctuated by tubulopapillary proliferations of squamoid cells and ciliated columnar cells. The Warthin-like MECs were comprised of cystic spaces lined by multilayered oncocytic to squamoid cells surrounded by a circumscribed cuff of lymphoid tissue with germinal centers. In these cases, the Warthin-like areas dominated the histologic picture. Conventional MEC, when present, represented a minor tumor component. MAML2 rearrangements were identified in all cases. Warthin-like MEC, and now a ciliated form of MEC, are newly described variants of a common salivary gland carcinoma. Unfamiliarity with these novel forms, unanticipated cellular features (eg, cilia), and morphologic overlap with mundane benign processes (eg, developmental ciliated cysts, Warthin tumor) or other carcinomas (eg, ciliated human papillomavirus-related carcinoma) may render these variants susceptible to misdiagnosis. These unusual variants appear to consistently harbor MAML2 fusions-a finding that establishes a clear link to conventional MEC and provides a valuable adjunct in establishing the diagnosis.

NOTCH1 is frequently mutated in adenoid cystic carcinoma (ACC). To test the idea that immunohistochemical (IHC) staining can identify ACCs with NOTCH1 mutations, we performed IHC for activated NOTCH1 (NICD1) in 197 cases diagnosed as ACC from 173 patients. NICD1 staining was positive in 194 cases (98%) in 2 major patterns: subset positivity, which correlated with tubular/cribriform histology; and diffuse positivity, which correlated with a solid histology. To determine the relationship between NICD1 staining and NOTCH1 mutational status, targeted exome sequencing data were obtained on 14 diffusely NICD1-positive ACC specimens from 11 patients and 15 subset NICD1-positive ACC specimens from 15 patients. This revealed NOTCH1 gain-of-function mutations in 11 of 14 diffusely NICD1-positive ACC specimens, whereas all subset-positive tumors had wild-type NOTCH1 alleles. Notably, tumors with diffuse NICD1 positivity were associated with significantly worse outcomes (P=0.003). To determine whether NOTCH1 activation is unique among tumors included in the differential diagnosis with ACC, we performed NICD1 IHC on a cohort of diverse salivary gland and head and neck tumors. High fractions of each of these tumor types were positive for NICD1 in a subset of cells, particularly in basaloid squamous cell carcinomas; however, sequencing of basaloid squamous cell carcinomas failed to identify NOTCH1 mutations. These findings indicate that diffuse NICD1 positivity in ACC correlates with solid growth pattern, the presence of NOTCH1 gain-of-function mutations, and unfavorable outcome, and suggest that staining for NICD1 can be helpful in distinguishing ACC with solid growth patterns from other salivary gland and head and neck tumors.