Immunohistology of Head and Neck Lesions

Reactive Changes

Reactive epithelial changes can have a variety of histologic patterns and cytomorphonuclear features. However, no immunohistochemical stains help identify or classify reactive changes, including pseudoepitheliomatous hyperplasia (PEH), or reliably distinguish them from dysplasia or neoplasia.

Dysplasia And Conventional Squamous Cell Carcinoma

SCC is the most common malignancy that arises in the head and neck. Invasive SCC tends to occur in the sixth decade or later and generally has a strong male predominance. Carcinogenesis is directly related to tobacco and/or alcohol in the vast majority of cases, ^, while a viral etiology for specific tumor types is well recognized. Epstein-Barr virus (EBV) is related to Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma (NPC) (see “Nasopharynx” section), and even smooth muscle tumors. There is a strong association between HPV and oropharyngeal carcinoma, squamous papilloma, and selected other anatomic sites carcinomas. ^{, , ,} p ^16ink4a may be used as a surrogate marker for transcriptionally active high-risk HPV in the oropharynx, but the p16 gene may also function as a tumor suppressor gene, and therefore altered expression does not absolutely correlate with the presence of HPV. Various techniques may be used (immunohistochemistry, in situ hybridization, PCR-based assays), but newer techniques (E6/E7 mRNA detection by ISH) offer greater promise in confirming transcriptionally active high-risk HPV.

When patients present with metastatic SCC in the upper neck lymph nodes, often a primary tumor cannot be identified. In these cases, p16 or in situ hybridization for HPV can be useful, as many of these tumors are from the tonsil and tongue base area. ^{, ,} When HPV is positive in the neck nodes from a patient with an unknown primary tumor, the clinical management can be directed toward these high-risk areas.

Dysplastic and neoplastic transformation of the squamous mucosa is typically classified into four basic categories: mild dysplasia, moderate dysplasia, severe dysplasia/carcinoma in situ (CIS) , and invasive carcinoma, although the World Health Organization now advocates low and high grade for lesions of the larynx. The cytologic and architectural features of dysplasia are quite characteristic, although with high inter- and intra-observer variability. In general, dysplasia shows architectural disorganization, thickening of the parabasal zones, lack of maturation with irregular perpendicular to parallel rotation, loss of polarity, bulbous or pointed rete, abnormal keratinization, even spongiosis, increased mitoses above the parabasal zone, and atypical mitoses. The cells show a similar size to the basal zone, have abnormal keratinization (glassy cytoplasm, dyskeratosis, karyorrhectic keratinization, surface keratinization, paradoxical keratinization), nuclear pleomorphism, nuclear hyperchromasia, nuclear irregularities, and nuclear streaming. Separation into mild, moderate, and severe dysplasia/CIS is based on progression of these features to involve the whole epithelium, although certain features (atypical mitoses, full thickness pleomorphism, absent maturation), when present anywhere in the epithelium, may still qualify the lesion as high-grade dysplasia.

Mutations and over-expression of the p53 gene are common in head and neck SCC, with approximately 50% to 60% of tumors showing aberrant p53 expression, ^{, , ,} although prognostic significance is not proven. However, p53 status may be linked to a response to chemo- and radiation therapy in SCCs. ^{, ,}

The intensity and location of both p53 and Ki-67 immunoreactivity are not helpful in dysplasia grading, while cyclinD1 over-expression, p16 deletion, loss of fragile histidine triad gene expression, and telomerase activity reactivation are potentially associated with dysplasia development or progression. ^, While epidermal growth factor receptor (EGFR) has several FDA-approved therapies that use this receptor for targeted drug therapy (such as cetuximab), over-expression by immunohistochemistry or somatic mutational analysis in tumors of the head and neck have met with only limited success.

In poorly differentiated tumors, particularly in metastatic sites, cytokeratin stains may be helpful since conventional SCC can usually be diagnosed on H&E alone. Typically, head and neck SCCs are positive for cytokeratin cocktails, AE1/AE3, pancytokeratin, and OSCAR. Cytoplasmic expression of keratins CK5, CK5/6, CK14, and CK17 are also frequently found in SCCs, as is nuclear p63 and p40 expression. ^{, ,} Table 9.1 highlights the patterns of cytokeratin reactions in various upper aerodigestive tract mucosal primary tumors.

Detection of metastatic disease may occasionally require the use of immunohistochemical stains in challenging specimens such as post radiation lymph nodes. Subtle post-radiation treatment residual tumors (primary or in lymph nodes) may show only granulomatous or necrotic tissue without viable tumor. In these cases, cytokeratin stains can be helpful to identify tumor within the necrotic deposits. Sentinel lymph node examinations are unreliable in head and neck tumors as there is a complex lymphatic drainage for most primary mucosal sites.

Oropharyngeal Squamous Cell Carcinoma

OPSCC is a histologically distinct type of SCC, arising from a specific anatomic site (base of tongue, lingual and palatine tonsils, hypopharynx, soft palate). ^{, , ,} The characteristic basaloid nonkeratinizing appearance is seen in many other locations, but does not have an overwhelming strong HPV association. ^{, ,} Classified as “HPV associated” or “HPV independent” OPSCC, the HPV associated tumors show a strong male greater than female bias, strong association with oral sex, and tend to develop in younger patients more than conventional SCC. ^{, ,} OPSCC may show nonkeratinizing , nonkeratinizing with maturation , and keratinizing histologic types. ^{, ,} There is a biphasic appearance, with the basaloid component dominant with only limited areas of keratinization or squamous differentiation ( Fig. 9.1 ). ^{, , ,} In contrast, classical basaloid SCC usually grows in smooth contoured lobules, large nests, or trabecular cordlike arrangements of small clusters or single cells. The lobules frequently contain central comedonecrosis with a peripheral palisade of nuclei. Cystic spaces and abortive ducts can be seen, along with a significant portion of cases showing basement membrane type material deposition and a cribriform growth pattern. Conventional SCC may be represented by focal dyskeratosis, keratin pearl formation, focal areas of “maturation,” invasive or in situ tumor, found separately or merged with the basaloid component ( Fig. 9.2 ). Cytologically, the cells are round to oval and have hyperchromatic nuclei with a high nuclear-to-cytoplasmic ratio. There are often prominent mitoses and apoptotic bodies. ^{, , , ,} The nonkeratinizing type has a very strong association with HPV no matter what detection method is used ( Fig. 9.3 ), slightly less so with the nonkeratinizing with maturation type and a weak association with the keratinizing type. ^, Transcriptionally active HPV is found in up to 97% of nonkeratinizing type SCC (see Fig. 9.3 ). ^{, , ,} However, there must be (1) a diffuse (>70% of the cells), strong, nuclear, and cytoplasmic block-type immunoreactivity in the neoplastic cells with p16 (see Fig. 9.3 ), or (2) a greater than 50% strong and diffuse staining combined with confluent staining (back-to-back cell staining of >25%) as surrogate markers of HPV status, and thus predictive of a better prognosis than HPV-negative tumors. ^{, ,} Interestingly, it seems that HPV+/p16+ tumors tend to have a better prognosis, although HPV-associated neuroendocrine carcinomas (NECs) may not show this association. By contrast, basaloid SCC (basaloid squamous cell carcinoma [BSCC]) of the larynx and sinonasal tract has a male predominance (M > F 4:1) and the mean age at diagnosis is about 63 years (range 27 to 88 years), associated with a worse prognosis than conventional SCC. ^{, , , ,}

BSCCs are positive for AE1/AE3, epithelial membrane antigen (EMA), CK5/6, CAM5.2, p63, p40, and SOX10, sometimes with carcinoembryonic antigen (CEA) (∼50%) and S100 protein (∼40%), but rarely with CD117. BSCCs are negative for neuroendocrine markers ( Fig. 9.4 ). The pattern of distribution of p63/p40 between the strong and diffuse reaction in BSCC versus the peripheral “myoepithelial/basal” staining in adenoid cystic carcinoma (ACC) may help separate between these tumors. ^{, ,} p53 is often strongly positive in BSCC, while only rarely seen in solid type or in high-grade ACC. ^{, , ,}

The differential diagnosis for OPSCC is broad, especially in small biopsies where all of the histologic features may not be appreciated. The major differential includes ACC, sinonasal undifferentiated carcinoma (SNUC), NPC (non-keratinizing type), small cell NEC, and NUT carcinoma. Immunohistochemical reaction differences between these tumors, combined with their unique anatomic sites and histologic features, are useful in resolving this differential diagnosis (see Table 9.1 ).

Spindle Cell Squamous Cell Carcinoma

Spindle cell “sarcomatoid” squamous cell carcinoma (SCSCC) is a rare variant of SCC showing a prominent to exclusive spindled to pleomorphic cell population. The tumor usually affects elderly males much more commonly than females (male:female = 12:1). Common primary locations of tumors include the glottis (70%), supraglottis, and numerous other head and neck locations. Tobacco and alcohol are the leading risk factors.

Grossly, the vast majority of tumors are polypoid (mean, 2 cm), often with an ulcerated surface. Histologically, SCSCC can be quite difficult to diagnose, particularly on small biopsies. The bulk of the tumor is composed of the spindled component, ranging from hypo- to hypercellular, and from bland to highly atypical ( Fig. 9.5 ). An overlying dysplastic (see Fig. 9.5 ), or more rarely, an invasive SCC component can help suggest the true nature of the tumor.

Immunohistochemistry may aid in confirming the diagnosis, but it is important to realize that up to 30% of cases will be negative with keratins. ^, In general, if keratins are going to be positive in the spindle cells, the best result is with pan-keratin (AE1/AE3/CK1), CAM5.2, or OSCAR, while EMA, p63, or p40 are also variably positive ( Figs. 9.6 and 9.7 ). Vimentin is always positive (see Fig. 9.6 ), but CK20 and HMB45 are uniformly negative. SCSCC can show variable expression with S100 protein (5%), actin-SM (30%), actin-HHF-35 (15%), CK5/6 (7%), CK14 (15%), CK17 (15%) (see Fig. 9.7 ); a positive result with aberrant markers should not be used to exclude the diagnosis. ^{, ,}

The major differential diagnosis for SCSCC includes reactive and benign neoplastic stromal proliferations and rare primary sarcomas of mucosal sites. Benign entities include granulation tissue, lobular capillary hemangioma (LCH), contact ulcer, leiomyoma, fibromatosis, and nodular fasciitis. Malignant neoplasms include melanoma, fibrosarcoma, synovial sarcoma, leiomyosarcoma, biphenotypic sinonasal sarcoma (BSS), among others. Immunohistochemistry can be helpful in differentiating SCSCC from some of these lesions, but because of overlapping histologic and immunophenotypes, the final diagnosis will rest on the histologic features and a compatible immunoprofile. Interestingly, SCSCC that lack immunohistochemical staining for epithelial markers have a better prognosis.

Olfactory Neuroblastoma

ONB is the prototypic “small, round, blue cell tumor” of the sinonasal tract. It comprises about 3% of all sinonasal tract tumors, arising from the specialized sensory neuroepithelial (neuroectodermal) olfactory cells that are normally found in the upper part of the nasal cavity, including the superior nasal concha, the upper part of the septum, the roof of the nose, and the cribriform plate of the ethmoid sinus. Thus, ectopic primaries must be viewed with skepticism. The normal olfactory epithelium contains three cell types, which can be histologically identified in the tumorous counterpart: basal cells, olfactory neurosensory cells, and supporting sustentacular cells. Transcriptomic and proteomic data have suggested a molecular separation of ONB into basal or neural type based on IDH2 R172 mutant-enriched tumors versus loss of DNA methylation at the enhancers of axonal guidance genes. ONB has a peak in the fifth and sixth decades, although it can present over a broad age range (2 to 94 years), affecting males slightly more often than females (1.2:1). ^, The tumors are frequently polypoid, ^, with a lobular architecture irrespective of tumor grade, comprised of “primitive” neuroblastoma cells. The circumscribed lobules or nests of tumor are below an intact mucosa and separated by a vascularized fibrous stroma ( Fig. 9.8 ). Rosettes, Homer Wright (∼30% of cases), and Flexner-Wintersteiner (5%) may be seen. Necrosis and mitoses are only seen in grade 3 and 4 tumors. The tumor cells are “small, round, blue” cells, slightly larger than mature lymphocytes, arranged in a syncytium with a high nuclear to cytoplasmic ratio. The nuclei are homogeneously uniform, with hyperchromatic, delicate, uniform “salt-and-pepper” chromatin. The background is formed from a tangle of neuronal process, giving a fibrillar quality. The nuclear features become more pleomorphic, and there is less stroma as the grade of the neoplasm increases (grade I to IV), with more of a reason to perform immunohistochemistry. ^{, ,}

Immunohistochemistry is more helpful when the tumor is higher grade, as the differential diagnosis is broader ( Table 9.2 ). A panel approach aids in diagnosis: pancytokeratin, p40, S100 protein, SOX10, synaptophysin, desmin, CD99, INI1, and CD45RB will provide the most meaningful initial results, which can then be selectively added to based on histologic features and other parameters to reach a diagnosis. The tumor cells are positive for neuroendocrine markers, including synaptophysin, chromogranin, INSM1, ISL-1, and even TTF1 ( Fig. 9.9 ). ^, The sustentacular supporting cells (Schwannian) at the periphery of the lobules are reactive with S100 protein, SOX10, and/or glial fibrillary acidic protein (GFAP) ( Fig. 9.10 ). ^{, , ,} About 5% of tumors may show a focal, cytoplasmic or membranous low-molecular-weight cytokeratin (CAM5.2) reaction, but it should not be diffuse or strong, and is generally not in a dot-like pattern. Calretinin (nuclear and cytoplasmic), neuron-specific enolase (NSE), ^, NKX2.2, neurofilament protein (NFP), ^{, ,} somatostatin receptor 2a (SSR2a), and hASH1 ^, are expressed in ONB to a variable degree, but other markers are more meaningful. INI1 is intact. Important negative immunohistochemistry antibodies include EMA, CEA, EBER, desmin, myogenin, p63, FLI1, CD99, CD117, HMB45, and CD45RB. PAX5 is rarely expressed but seems to be seen in tumors that behave aggressively. ^{, , ,} Rare cases may show divergent differentiation, including ganglion cells, melanin-containing cells, and rhabdomyoblasts; these component cells stain accordingly, but are focal findings. ^,

The “small round blue cell” tumor differential is considered within this spectrum and is discussed with each of the subsequent entities. While lymphoma is not covered in this chapter, NK/T-cell nasal type, B-cell lymphomas, and plasmacytoma are considered within the differential diagnosis of ONB.

Sinonasal Undifferentiated Carcinoma

SNUC is an aggressive malignant epithelial neoplasm without evidence of squamous or glandular differentiation. As a diagnosis of exclusion, SMARCB1-deficient carcinoma, SMARCA4-deficient carcinoma, NUT carcinoma, and ALES should be recognized and excluded, noting they are frequently reactive with squamous markers. SNUC is locally destructive, associated with necrosis, and must be separated from many tumors in this site. SNUC is more common in men than women (3:1) and occurs over a broad age range (mean, sixth decade). The tumor arises within the nasal cavity, but quickly spreads to the paranasal sinuses, orbits, and skull base. The tumors are large (mean, >4 cm), fungating, with ill-defined margins.

Microscopically, SNUC shows several patterns of growth, including lobular, trabecular, sheet-like, ribbons, and solid islands. Confluent tumor necrosis and comedonecrosis are prominent; bone destruction, lymphovascular invasion, and perineural invasion are easily identified ( Fig. 9.11 ). Surface ulceration obscures surface involvement, which is very rare. The polygonal cells are monotonously medium to large, with a high nuclear to cytoplasmic ratio, hyperchromatic to vesicular nuclear chromatin, easily identified nucleoli, and usually a syncytial appearance (see Fig. 9.11 ). No neurofibrillary matrix or true rosettes are present.

SNUCs are almost always strongly and diffusely positive for CK-pan, OSCAR, CK8, CAM5.2 (see Fig. 9.11 ), and p16 ( Table 9.2 ), while positive with CK7, CK19, EMA, and p53 in about 50% of cases (see Fig. 9.11 ). p63 is positive in a few cases, but p40, CK5/6, CK4, and CK14 are nonreactive. ^{, ,} A few cases may also show neuroendocrine marker reactivity, along with CD99 and S100 protein (<15%), although purists argue that SNUC should only be applied to “undifferentiated” tumors. CD117 is positive, while calretinin, NKX2.2, muscle markers, melanocytic markers, EBER, NUT, and hematolymphoid markers are negative. ^, Curiously, most cases are strongly positive with p16, but lack any HPV DNA expression. ^, Tumors may be IDH -mutated, with IDH protein expression in the cytoplasm.

It may be difficult to separate SNUC from NPC, small cell NEC, NUT carcinoma, SMARCB1 -deficient carcinoma, or SMARCA4 -deficient based on histology alone. In most cases, SNUC is usually negative with CK5/6, p63, and CK903, while NPC is strongly positive with EBER, CK5/6, CK903, and p63. ^{, ,} NUT carcinoma is positive with the NUT antibody (nuclear), CK5/6, p40, and p63. ^{, ,} Small cell NEC must show histologic neuroendocrine differentiation, while tending to show a punctate or dot-like CK-pan and CAM5.2 reaction, and neuroendocrine marker reactivity. The SMARCB1 (INI1) deficient carcinoma and SMARCA4 -deficient carcinoma groups generally lack keratinization and may show plasmacytoid cells. By definition, due to biallelic inactivation of SMARCB1 , there is a loss of INI1 nuclear immunoexpression, with BRG1 loss in SMARCA4 -deficient tumors, while IDH immunohistochemistry may aid in confirming SNUC. ^,

Nut Carcinoma

NUT carcinoma is an aggressive SCC subtype defined by NUTM1 (NUTM1) gene rearrangements (see Table 9.2 ). Patients are usually young, with a slight female predominance, presenting with a rapidly growing, often midline mass involving the sinonasal tract and orbit.

Histologically, the tumor shows an undifferentiated or primitive sheet-like distribution of a poorly differentiated SCC that are often quite monotonous. The cells have a slightly blue to clear cytoplasm surrounding vesicular to delicate nuclear chromatin. Areas of abrupt keratinization or squamous differentiation are very helpful in the diagnosis ( Fig. 9.12 ). Areas of necrosis and acute inflammation may be seen.

The neoplastic cells are immunoreactive with NUT (see Fig. 9.12 ), CK-pan, p63, p40, CK5/6, p16, and occasionally with neuroendocrine markers. ^{, , ,}

The differential diagnosis includes poorly differentiated SCC, SNUC, SMARCB1 -deficient carcinoma, ES, ALES, leukemia, and germ cell tumors.

Mucosal Melanoma

MMs develop from neural crest melanocytes found within the sinonasal tract, eye, oral cavity, and larynx. Within the sinonasal tract, they are uncommon, typically presenting in the fifth to eighth decades without a sex predilection. Most tumors arise on the nasal septum, but expand into the paranasal sinuses early, with a mean size of about 3 cm.

Surface ulceration may obscure junctional melanocytes, while amelanotic, spindled, epithelioid, or solid tumors require a high index of suspicion for diagnosis. The tumor may be solid, organoid, nested, storiform, papillary, fascicular, meningothelial, or distinctly peritheliomatous ( Fig. 9.13 ). The cells are protean, ranging from undifferentiated, polygonal, epithelioid, small cell, and giant cell, to plasmacytoid and rhabdoid ( Fig. 9.14 ). The cells will frequently have prominent, brightly eosinophilic nucleoli, intranuclear cytoplasmic inclusions, and opacified cytoplasm. Melanin is helpful when present. Mitoses are usually easily identified, as is tumor necrosis. ^{, , ,}

In general, S100 protein, SOX10, HMB45, tyrosinase, Melan-A (MART-1), MITF, PRAME (PReferentially expressed Antigen in MElanoma), and KBA.62 are variably positive in MM, with SOX10 perhaps the most sensitive and HMB45 the most specific ( Figs. 9.15 and 9.16 ; see Table 9.2 ). In addition, vimentin (100%), FLI-1 (up to 100% of cases), NSE (focal, <50% of cells), CD117 (∼33% of cases), CD99 (∼25% of cases), and even CD56 (<10% of cases) are variably positive. ^{, ,} Rarely, CAM5.2 and EMA may show focal positivity. In general, two positive melanocytic stains are more reassuring than only one positive reaction, depending on the morphology and clinical setting. Mutations in KIT and NRAS genes are much more common than BRAF in MM, with TP53 mutations frequently documented.

The differential diagnosis for MM is quite broad, depending on pattern and cytologic features, and includes ONB, SNUC, RMS, peripheral nerve sheath tumor (PNST; benign or malignant), leiomyosarcoma, melanotic neuroectodermal tumor of infancy, meningioma, plasmacytoma, and mesenchymal or myxoid chondrosarcoma, among others. ^{, ,} In general, a pertinent IHC panel can be ordered based on the anatomic site, age of the patient, and pattern of growth, and further honed by the specific location of the immunohistochemical reaction. As an example, ONB shows a sustentacular S100 protein reaction which would not be seen in MM. A PNST would be positive with S100 protein and SOX10, but would not show HMB45, Melan-A, or tyrosinase reactions ( Fig. 9.17 ). Mesenchymal chondrosarcoma also shows S100 protein reactivity, but is positive with SOX9, while negative with HMB45, Melan-A, or tyrosinase ( Fig. 9.18 ). ^{, ,}

Sinonasal Neuroendocrine Carcinoma

Several tumors in the sinonasal tract have “neuroendocrine” differentiation (SNUC, ONB, pituitary neuroendocrine tumor, paraganglioma, and NEC), but the diagnosis of a NEC should only be rendered when there are histologic neuroendocrine features combined with immunohistochemistry findings, and these other tumor types are actively excluded. Small cell and large cell NEC are histologically similar to their lung counterparts (in this section, the small cell carcinoma type is considered). The tumors develop in mid to older age men. The tumor involves the nasal cavity and/or the paranasal sinuses, frequently showing extension into adjacent structures. ^,

Microscopically, NEC shows sheets, ribbons, and nests of small cells with a high nuclear to cytoplasmic ratio. There is usually no overlying dysplasia or carcinoma. The cells may be round to spindled, with scant cytoplasm surrounding hyperchromatic nuclei with inconspicuous nucleoli. Nuclear molding, frequent mitoses, necrosis, and single-cell apoptosis are common ( Fig. 9.19 ).

NECs, specifically small cell carcinoma, must show neuroendocrine histologic features and immunohistochemistry findings that show a characteristic strong reaction for CK-pan and CAM5.2, usually in a punctate or dot-like pattern ( Fig. 9.20 ) (see Table 9.2 ). Cells are positive for p16 and EMA, focally with p63, while negative with CK5/6, CK20, EBER, and CD99 reactions (see Fig. 9.20 ). The diagnosis is confirmed by staining with at least one neuroendocrine marker, including synaptophysin, chromogranin, INSM1, ISL-1, CD57, NSE, and human achaete-scute homolog 1 (see Fig. 9.20 ). In some cases, the cells may be positive with S100 protein, CD117, TTF-1 (see Fig. 9.20 ), and brachyury. , , , , , , – CD56 is expressed, but is not a specific neuroendocrine marker. In rare cases, ectopic hormones expression (calcitonin, adrenocortical hormone, beta melanocyte stimulating hormone, serotonin, parathyroid hormone) may be detected. There is usually a high Ki-67 proliferation index, with consistent loss of RB1 and over-expression of p53 by IHC.

Metastatic disease to the sinonasal tract from a pulmonary primary must always be considered, along with SNUC, ES/ALES, pituitary neuroendocrine tumor (PitNET), and NUT carcinoma. Further, focal immunohistochemical neuroendocrine differentiation can be seen in other carcinomas as well as ONB and even RMS. PitNET does not have vascular invasion, lacks atypical mitoses, and will be reactive with various pituitary hormones or transcription factors, separated further by SF1, TPIT, or PIT1 reactivity. ES usually shows CD99, NKX2.2, ERG, and FLI1, while only showing weak to focal keratin and neuroendocrine reactions. ^, The ALES shows strong keratin, p40, and NKX2.2 reactivity. RMS can express neuroendocrine markers, but also shows myogenic markers. NUT carcinoma must express NUT immunohistochemistry, a marker quite specific and unique to this neoplasm.

Pituitary Neuroendocrine Tumor (Pituitary Adenoma)

PitNET is the preferred term for pituitary adenoma in an attempt to harmonize neuroendocrine tumor terminology universally. It may develop in the sinonasal tract by direct extension from an intracranial tumor (most common, in up to 3% of cranial primaries) or as an ectopic tumor which has a normal sellar and tumor in the sphenoid sinus, followed by nasopharynx and nasal cavity. These tumors are frequently misdiagnosed as other neoplasms in these sites. The tumors usually present in the sixth decade (mean, 54 years) with a slight female to male bias (1.3:1). Hormone production may result in clinical symptoms or serologic hormone elevation in some cases. Tumors are often large (mean, 3 cm).

Tumors are identified beneath an intact respiratory epithelium, arranged in many different patterns (solid, packets, organoid/insular, rosettes, single file, glandular, trabecular; Fig. 9.21 ), often with bone invasion but lacking lymphovascular invasion. Secretions are common; necrosis is infrequently present. Mitoses may be seen but without atypical forms. The tumors show a variable cellularity, with polygonal, plasmacytoid, granular, and oncocytic tumor cells. Severe pleomorphism is uncommon. The nuclear chromatin is delicate, with a salt-and-pepper distribution (see Fig. 9.21 ), showing intranuclear cytoplasmic inclusions. The cells are supported in a vascularized to sclerotic stroma.

Immunohistochemistry highlights the neuroendocrine nature, with synaptophysin (>95%), NSE (>75%), chromogranin (>70%), and INSM-1 identified in a variety of distributions (membrane, cytoplasmic, dot-like), with CK-pan (80%) or CAM5.2 (60%) confirming the epithelial component, often in a dot-like Golgi accentuation ( Figs. 9.22 and 9.23 ) (see Table 9.2 ). Up to 60% of cases are positive for pituitary hormones: prolactin (most common), follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, thyroid stimulating hormone, and growth hormone, often plurihormonal ( Fig. 9.24 ). If required, pituitary transcription factors (such as Pit-1, TPIT, SF-1, ER-α, GATA-2) will help to confirm the diagnosis in hormone silent cases. Calcitonin is rarely positive; CK7, CK5/6, p63, p40, TTF1, SOX10, HMB45, EBER, and S100 protein are usually negative. ^{, ,}

The differential diagnosis includes ONB, NEC, SNUC, paraganglioma, ES, ALES, and MM. The relative monotony of the cells, lack of lymphovascular invasion, no atypical mitoses, and tumor location help to guide the immunohistochemistry evaluation (i.e., pituitary hormones would be performed after a lymphoma, RMS, or melanoma were excluded).

Rhabdomyosarcoma

RMS is the most common soft tissue sarcoma of children and is also the most common soft tissue sarcoma in the head and neck. Within the head and neck, RMS primarily involves the orbit, ear and temporal bone, and sinonasal tract/oro- and naso-pharynx. ^{, ,} Embryonal RMS tends to develop in the ear/mastoid, while alveolar RMS is more common in the sinonasal tract ( Fig. 9.25 ). Specifically, in the sinonasal tract, alveolar RMS develops in adults with a slight male predominance (male:female = 1.2:1). ^{, , ,}

In the sinonasal tract, the tumors are below an intact or partially ulcerated epithelium. The primitive mesenchymal cells are arranged in sheets to loose alveolar patterns, with cells loosely attached to the periphery of the nest, showing central degeneration or dilapidation (see Fig. 9.25 ). Focal tumor cell spindling (see Fig. 9.25 ) or plasmacytoid/rhabdoid appearance may be seen. The cells have a high nuclear to cytoplasmic ratio, slightly eccentric, darkly eosinophilic cytoplasm around pleomorphic, hyperchromatic nuclei. Multinucleation is common, as are mitoses. Cross striations are rare. Necrosis is present. ^{, , ,}

RMS is positive with vimentin, desmin, myoglobin, and myosin, with strong nuclear reactions for myogenin, MITF, and MYOD1 ( Figs. 9.26 and 9.27 ). ^{, , , ,} Myogenin tends to be stronger, especially in cells adjacent to fibrous septae, in alveolar RMS than in embryonal RMS. The tumor cells are also positive with actins and CD56, while up to 30% of cases will show a weak, focal, punctate or dot-like CK-pan and/or CAM5.2 immunoreactivity (see Fig. 9.27 ). ^{, ,} S100 protein and HMB45 are negative, but there may be weak to focal reactivity with synaptophysin, chromogranin, NSE, CD99, PAX2, and FLI1. Alveolar RMS has a characteristic translocation between the FOXO1 (13q14) forkhead region and either PAX3 (2q35) or PAX7 (1p36), which can be confirmed by FISH for FOXO1 or by RT-PCR. ^{, ,}

Although the other “small, round, blue cell tumors” of the sinonasal tract are included in the differential diagnosis as presented in Table 9.2 , it is important to remember several unique sinonasal tract tumors in the differential diagnosis, which may have rhabdoid or rhabdomyoblastic features. ONB may have rhabdoid differentiation. ^, Teratocarcinosarcoma is a unique sinonasal tract malignancy that shows a teratoma-like distribution of carcinoma and sarcoma, the latter frequently a RMS ( Fig. 9.28 ). Malignant peripheral nerve sheath tumors (MPNSTs) may show rhabdomyoblastic differentiation, frequently referred to as a “Triton tumor” ( Fig. 9.29 ). Desmoplastic round cell tumor is a unique entity, which shows desmin, keratin, neuroendocrine marker, and WT1 reactivity, but is negative with myogenin, MYOD1, and S100 protein ( Fig. 9.30 ); a FISH or RT-PCR for the EWSR1 / WT1 translocation (t(11;22)(p13;q12—the classic translocation) is confirmatory. ^, Caution is advised when entrapped or atrophic muscle fibers may be within a tumor and may react with myoid markers.

Ewing Sarcoma And Adamantinoma-Like Ewing Sarcoma

ES is a high-grade primitive small round cell tumor sarcoma with neuroectodermal differentiation and defined by the EWSR1 gene translocation, most common with FLI-1 . An adamantinoma-like EW (ALES) may be seen in the head and neck (salivary gland, sinonasal tract), where there is squamous differentiation and strong p40 expression. The Ewing family of neoplasms may be found in bone, soft tissue, and various parenchymal organs (lung, pancreas, kidney), with approximately 10% developing in the head and neck. ^{, ,} The Ewing family of tumors show several molecular alterations, but the majority result from a fusion of the EWSR1 gene with the FLI-1 or ERG genes, respectively, creating a chimeric gene product.

Approximately 80% of patients are less than 20 years old at presentation, with adults uncommonly affected, although adults are affected with ALES. In all sites, there is a slight male predilection (male:female = 1.4:1), but this is not as prominent in sinonasal tract tumors. Extension by bone destruction beyond the sinonasal tract to adjacent organs (orbit, brain) is common, resulting in a large tumor size at presentation (up to 6 cm). ^{, , , ,}

ESs are composed of uniform round cells, which grow in a sheet-like to lobular or nested configuration ( Fig. 9.31 ), frequently showing coagulative or geographic necrosis. Delicate vessels course through the proliferation, often compressed by the tumor cells. Mitoses are easy to identify. The cells have a high nuclear to cytoplasmic ratio, with finely dispersed to powdery nuclear chromatin with small to inconspicuous nucleoli. The cytoplasm is scanty, poorly defined, and frequently pale or cleared owing to an abundance of glycogen ( Figs. 9.31 and 9.32 ). Homer Wright pseudorosettes (up to 10% of cases) or rarely Flexner-Wintersteiner rosettes may be present. Atypical, spindled, large cell, clear cell, hemangioendothelioma-like, sclerosing, and adamantinoma-like variants are recognized. The adamantinoma-like variant has squamous differentiation and shows a strong reactivity with p40 ( Fig. 9.33 ). ^{, ,}

ES are strongly and diffusely reactive CD 99, usually in a membranous fashion (see Fig. 9.32 ); NKX2.2 (see Fig. 9.33 ), FLI1, ERG, SNF5, vimentin, and p16 are usually positive. ^{, , , ,} FLI1 protein nuclear expression is not specific for ES, although the EWSR1 / FLI-1 fusion gene is specific. ^, Tumor cells may also react with neuron-specific enolase (50%), synaptophysin (∼35%), claudin-1 (∼40%), CD117 (∼35%), S100 protein (30%), GFAP (20%), calretinin (15%), and CD56 (10%). Up to 30% may also be positive for CK-pan (see Fig. 9.33 ), CAM 5.2, and/or EMA, with strong p40 reactivity seen in ALES (see Fig. 9.33 ). ^{, , ,} Rarely, desmin and chromogranin (2%) may be focally present, while WT1 and myogenin are negative.

The principle differential diagnoses include ONB, lymphoma, RMS, mesenchymal chondrosarcoma, small cell osteosarcoma, pituitary neuroendocrine tumor, SCC, NUT carcinoma, basaloid salivary gland neoplasms, and SNUC. A pertinent and targeted panel will often resolve these cases to the correct diagnosis. However, it is critical to remember that significant overlap in immunohistochemical reactions can be seen, not only in positive versus negative, but also in location and character of the reaction.

There are three additional tumors of the sinonasal tract which deserve consideration, specifically because immunohistochemistry assists with accurate classification and diagnosis. They are sinonasal tract intestinal type and non-intestinal type adenocarcinomas, glomangiopericytoma (with differential diagnoses), and the unique biphenotypic sinonasal sarcoma (BSS).

Sinonasal Intestinal-Type Adenocarcinoma

Adenocarcinomas of the sinonasal tract are separated into three major groups by the World Health Organization: salivary gland type intestinal-type adenocarcinomas (ITACs), and non-ITACs. Specifically, it is the intestinal-type and non-intestinal type adenocarcinomas that are more frequently misinterpreted, with SMARCB1 -deficient variant, ETV6 gene rearranged, and IDH2 mutated tumors recognized within sinonasal tract adenocarcinomas. The ITAC group are rare tumors that recapitulate intestinal adenocarcinomas. There is a very strong epidemiologic link to occupational exposure, specifically in the furniture and shoe/leather industries, developing after a very prolonged exposure history, often to particulate matter. ^, ITAC is significantly more common in men than women (male:female = 4:1) and peaks in the fifth to seventh decades. In non-exposure cases, there is a slight female predominance. ^, If detected as part of a screening program, tumors tend to be much smaller than those encountered in non-exposure related patients. One of the major dilemmas in ITAC is differentiating them from metastatic adenocarcinoma of the colon, where accurate clinical history is critical. ^{, , , ,}

The Barnes classification is preferred and separates the tumors into five morphologic categories based on pattern and cytologic features: colonic (40%), solid (20%), papillary (18%), mucinous (8%), and mixed (12%) ( Fig. 9.34 ). In general, the colonic type has a tubuloglandular architecture, only rare papillae, and an increased nuclear pleomorphism and nuclear stratification with mitoses. The solid type is solid to trabecular with rare tubules, smaller cuboidal cells, vesicular nuclei, prominent nucleoli, and nuclear pleomorphism. The papillary type has a dominant papillary architecture (like a tubular-villous adenoma of the colon), showing limited pleomorphism and rare mitoses. The mucinous type shows mucin either intracellularly or in extracellular pools. Large glands may be distended by mucin, or there may be signet-ring type cells floating in mucin lakes. Mixed types have an admixture of any of these aforementioned types. No matter which type is present, villi, Paneth cells, enterochromaffin cells, and even a muscularis mucosa can be seen. Yolk-sac differentiation may be seen. Tumor budding, conceptually similar to colon, has been proposed as a prognostic marker.

ITACs show both sinonasal and intestinal immunohistochemical reactions. ITACs will give a strong reaction for CK7 ( Fig. 9.35 and Table 9.3 ), ^, while also strongly positive for CK20, CDX2, SATB2, MUC1, MUC2, typically seen in intestinal-derived malignancies (see Fig. 9.35 ). ^{, , , ,} Further, they will react with villin, MUC4, MUC5AC, and CEA-p (see Fig. 9.35 ), and a wide variety of neuroendocrine markers (synaptophysin, chromogranin) or even hormone peptides such as serotonin, gastric, or somatostatin. p53 is usually over-expressed, although to a lesser degree in the mucinous type. ^, Several other epithelial markers (EMA, B72.3) show variable reactivity. Loss of INI1 supports SMARCB1 -deficient adenocarcinoma. Tumors are negative with vimentin, actins, p63, HER2/neu (ERBB2), and CK903, ^, while IDH1/2 immunohistochemistry can be used when positive to confirm a mutation. Interestingly, the low levels of KRAS , EGFR , and BRAF mutations in ITACs suggest a different pathogenesis, while also suggesting difference in targeted therapies. ^, The different expressions of mucins are known to be associated with growth pattern, survival, and distant metastasis, as well as suggesting targets for new treatments. Genomic profiling shows distinct subgroups that relate to outcome but are independent of stage or histologic subtype. Gene fusion specific carcinomas are uncommon, but secretory carcinoma (SC) and the ETV6 -rearranged low-grade sinonasal tract adenocarcinoma may be such entities. ^{, ,}

In general, without a good clinical history, imaging studies, or colonoscopy, it may be impossible to separate a metastatic tumor from the GI tract to the sinonasal tract based on histology or immunohistochemistry alone. Colon carcinomas tend not to be CK7 positive, and only rarely are reactive with chromogranin and MUC5. Separation from sinonasal nonintestinal adenocarcinomas (SNACs) may be easier since they are nonreactive for CK20, CDX-2, villin, SATB2, and MUC2, although CEA and MUC5 may be positive. ^{, ,} Other lesions in the differential diagnosis may include malignancies (salivary gland adenocarcinomas, nasopharyngeal papillary adenocarcinoma [NPPA]), metastatic carcinomas, and benign conditions such as papillary rhinosinusitis, respiratory epithelial adenomatoid hamartoma ( Table 9.4 ), and seromucinous hamartoma. ^{, ,}

There is a SNAC referred to as “sinonasal renal cell-like carcinoma,” a tumor showing monomorphous cuboidal to columnar glycogen-rich clear cells that lack mucin production ( Fig. 9.36 ). They histologically mimic metastatic renal cell carcinoma (RCC), although the renal cell-like carcinoma shows CAIX and CD10, but are negative with PAX8 and RCC marker (see Fig. 9.36 ). There is a cohort of ETV6 -mutated sinonasal adenocarcinomas that are not SCs.

Glomangiopericytoma

Glomangiopericytoma was originally designated as sinonasal-type hemangiopericytoma, but the perivascular myoid phenotype is more closely related to glomus tumor. ^{, ,} Glomangiopericytoma is an uncommon soft tissue tumor affecting the nasal cavity more often than the paranasal sinuses. The tumor usually develops in the seventh decade, but shows a broad age range at presentation, with slight female predominance (female:male = 1.2:1). The tumors present with a polypoid mass, showing a mean size of about 3.0 cm.

Microscopically, there is a subepithelial proliferation separate from a usually intact surface epithelium. The tumor is arranged in short, compact to whorled or palisaded fascicles composed of spindled to epithelioid cells with indistinct cell borders ( Fig. 9.37 ). The cytoplasm is amphophilic surrounding nuclei with coarse nuclear chromatin and without pleomorphism. There is a well-developed, branching vascular stroma. Heavy peritheliomatous hyalinization is characteristic (see Fig. 9.37 ). Extravasated erythrocytes, mast cells, and eosinophils are noted throughout (see Fig. 9.37 ). Mitoses are inconspicuous. Tumor giant cells, lipomatous change, and hematopoiesis can be present, along with solitary fibrous tumor (SFT). Rare malignant cases are recognized. ^,

The neoplastic cells are reactive with vimentin, actins (smooth muscle > muscle specific), nuclear β-catenin ( Fig. 9.38 ), LEF1, nuclear cyclin D1, and factor XIIIa ^{, , , ,} while laminin highlights the matrix. Isolated tumor cells in a few cases are positive with CD34, S100 protein, desmin, bcl-2, CD68, and GFAP. CD34 expression is present when the Leica QBEND10 clone is used rather than the MUC-236-4C BioGenex clone (see Fig. 9.38 ). The tumor cells are negative with CD31 (see Fig. 9.38 ), ERG, D2-40, FVIIIRAg, CK-pan, CK7, EMA, SOX10, STAT6, TLE1, NSE, EBER, and CD117 (although the mast cells will be positive). ^{, , ,}

The differential diagnosis includes LCH, SFT, BSS, synovial sarcoma, sinonasal tract angiofibroma (SNAF), fibrosarcoma, PNST, desmoid fibromatosis, melanoma, fibrosarcoma, leiomyosarcoma, and meningioma ( Table 9.5 ). In general, the heavy inflammatory infiltrate and “granulation tissue-like” appearance in LCH helps make the separation, with cells positive for CD31, CD34, and FVIIIRAg. An SFT may be concurrently present with glomangiopericytoma, but as a differential consideration, usually shows a lower cellularity, a higher deposition of wiry collagen ( Fig. 9.39 ), and a strong CD34, STAT6, and bcl-2 reaction ( Fig. 9.40 ). STAF occurs in a different location, exclusively in males, usually before age 20 years, and shows a spectrum of vessels within the lesion. PNSTs may also have peritheliomatous hyalinization but tend to have areas of Antoni B myxoid degeneration and show strong S100 protein and SOX10 immunoreactivity. Meningiomas are usually meningothelial in this site, may have psammoma bodies, and tends to lack the rich vascularity of a glomangiopericytoma. Meningioma may be EMA and CK7 positive, but does not usually react with SMA, MSA, or Factor XIIIa. BSS tends to have entrapped epithelial islands, lacks an inflammatory component, and does not have a peritheliomatous hyalinization. There is a strong, although often focal reaction with S100 protein and SMA.

Biphenotypic Sinonasal Sarcoma

BSS is a low-grade spindle cell sarcoma of the sinonasal tract, showing a distinctive histologic, immunohistochemical, and molecular profile. Formerly, low-grade sinonasal sarcoma with neural and myogenic features, the tumor was previously diagnosed as a fibrosarcoma, malignant PNST, or leiomyosarcoma. Females are affected more often than males (2:1), over a wide age range (mean, 52 years), typically involving several sites within the sinonasal tract. Tumors are usually non-descript, up to about 4 cm in size.

The tumors are cellular, showing a subepithelial proliferation of spindled tumor cells in medium to long intersecting fascicles, often showing a herringbone pattern. Tumors often infiltrate the bone (about 20%) but tend to lack necrosis. There is limited pleomorphism, with tapering, slender nuclei within the spindled tumor cells. Mitoses are sparse. Characteristic is the entrapped respiratory epithelium, often showing invagination into the spindled cells, but occasionally showing gland-like profiles ( Fig. 9.41 ). Rhabdomyoblastic differentiation may be seen (see Fig. 9.41 ), interestingly, showing a different fusion partner by molecular analysis (usually NCOA1/2 or FOXO1 ). ^,

The neoplastic cells show variable focal to diffuse, weak to strong S100 protein and SMA reactivity, with nuclear β-catenin and PAX3 reactivity ( Fig. 9.42 ). ^, Isolated cells may show CD34, EMA, or pancytokeratin reactivity, while desmin, myogenin, and/or MYOD1 may be expressed in the rhabdoid areas (see Fig. 9.42 ); there is an absence of SOX10, STAT6, HMB45, and other vascular markers. ^, A highly expressed translocation (t(2;4)(q35;q31.1)) results in a fusion transcript between PAX3 and MAML3 , while PAX3 - FOXO1 and PAX3 - NCOA1/2 may also be seen. ^{, , ,}

The differential diagnosis includes synovial sarcoma, malignant PNST (including triton tumor), leiomyosarcoma, spindle cell RMS, MM, glomangiopericytoma, NTRK -rearranged spindle cell neoplasms, and SFT (see Table 9.5 ). The characteristic glandular/epithelial inclusion, limited pleomorphism, and mitoses helps in most cases, although synovial sarcoma is the most challenging, often requiring molecular studies to aid in diagnosis.