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

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 leiomyosarcoma. There is a strong association between HPV and oropharyngeal carcinoma, squamous papilloma, and selected other anatomic sites carcinomas. p16 ^ink4a 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 [ISH], 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 ISH for HPV can be useful, as many of these tumors are from the tonsil and tongue base area (oropharynx). 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 (WHO) now advocates low- and high-grade dysplasia for lesions of the larynx. The cytologic and architectural features of dysplasia are quite characteristic, although with high interobserver and intraobserver 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, still qualify the lesion as high-grade dysplasia.

Mutations and overexpression 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 chemotherapy and radiation therapy in SCCs.

The intensity and location of both p53 and Ki-67 immunoreactivity are not helpful in dysplasia grading, whereas cyclin D1 overexpression, p16 deletion, loss of fragile histidine triad gene expression, and telomerase activity reactivation are potentially associated with dysplasia development or progression. Although epidermal growth factor receptor (EGFR) has several US Food and Drug Administration (FDA)–approved therapies that use this receptor for targeted drug therapy (such as cetuximab), overexpression 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, along with nuclear p63 and p40 expression. Table 9.2 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 postradiation 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 in identifying tumor cells 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 (e.g., 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 a similar strong HPV association. Classified as “HPV-associated” or “HPV-negative” OPSCC, the HPV-associated tumors show a strong male to female bias, strong association with oral sex, and tend to develop in younger patients than those associated with 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 (BSCC) 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 some 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,” or 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 regardless of the detection method 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 ( Fig. 9.3 ). However, there must be (1) a diffuse (>70% of the cells), strong, nuclear, and cytoplasmic immunoreactivity in the neoplastic cells with p16 ( Fig. 9.3 ), or (2) a >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 (NEC) may not show this association. By contrast, BSCC of the larynx and sinonasal tract has a male predominance (4 : 1), the mean age at diagnosis is about 63 years (range 27 to 88 years), and it has a worse prognosis than conventional SCC.

Basaloid squamous cell carcinoma (A–D). The various features of tumor cell nests, focal inflammation, increased mitoses and lack of maturation are highlighted in this panel.

Oropharyngeal squamous cell carcinoma. (A) Isolated foci of keratinization in this sheet-like growth. (B) Focal area of keratinization (maturation) in an otherwise nonkeratinizing squamous cell carcinoma.

Oropharyngeal carcinoma, nonkeratinizing type. (A) p16 with a strong, diffuse nuclear and cytoplasmic reaction. (B) Human papillomavirus with dot-like nuclear reaction detected by RNA in situ hybridization.

BSCCs are positive for AE1/AE3, epithelial membrane antigen (EMA), CK5/6, CAM5.2, p63, and p40, 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 high-grade ACC.

Basaloid squamous cell carcinoma from the nasal cavity. (A) Basaloid neoplastic cells with central comedonecrosis and abrupt keratinization. (B) CK-pan shows strong cytoplasmic reaction in the neoplastic cells. (C) CK5/6 highlighting the inner cells more strongly than the outer basaloid cells. (D) p63 highlights the basaloid cells at the periphery in this example of adenoid cystic carcinoma.

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 (nonkeratinizing type), small cell neuroendocrine carcinoma (SCNEC), and nuclear protein in testis (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 9.2 ).

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 men much more commonly than women (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 hypocellular to hypercellular, and from bland to highly atypical ( Fig. 9.5 ). An overlying dys­plastic 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 keratin negative. 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 ( Fig. 9.6 ), but CK20 and HMB-45 are uniformly negative. SCSCC can show variable expression with S100 protein (5%), actin-SM (30%), actin-HHF-35 (15%), CK5/6 (7%), and CK14 (15%), and CK17 (15%; Fig. 9.7 ). A positive result with aberrant markers should not be used to exclude the diagnosis.

Spindle cell squamous cell carcinoma. (A) The spindled cell population is immediately adjacent to an area of frank squamous cell carcinoma, showing a keratin pearl formation. (B) This spindle cell proliferation shows remarkable pleomorphism with a large number of mitoses, including many atypical forms.

The neoplastic spindle cells of a spindle cell squamous cell carcinoma are highlighted by a variety of different markers. (A) CK-pan; (B) vimentin; (C) epithelial membrane antigen; (D) CK18.

The neoplastic spindle cells of a spindle cell squamous cell carcinoma highlighted by a variety of different markers. (A) CK14; (B) CK1; (C) CK17; (D) focal, but strong, smooth muscle actin.

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, SCSCCs 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. 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, composed 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).

Olfactory neuroblastoma (ONB). (A) The lobular architecture is maintained irrespective of the grade of tumor. (B) Small pseudorosettes are noted in this lobule of ONB. (C) The primitive cells show a high nuclear-to-cytoplasmic ratio. Note the intact respiratory epithelium overlying. (D) Dystrophic calcifications within a well-vascularized stroma between lobules of tumor.

Immunohistochemistry is more helpful when the tumor is higher grade, as the differential diagnosis is broader ( Table 9.3 ). The tumor cells are positive for neuroendocrine markers, including synaptophysin, neuron-specific enolase (NSE), CD56, neurofilament protein (NFP), and chromogranin ( Fig. 9.9 ).There is often a strong and focal to diffuse (>75% of cells) reactivity with calretinin ( Fig. 9.10 ). Isolated cells may be positive with CK-pan or CAM5.2 (up to 30% of cases) but are negative with EMA, muscle markers, CD99 (MIC-2), HMB-45, CD117, and EBER. Rare cells may be positive with p63. The sustentacular support cells (schwannian) at the periphery of the lobules are reactive with S100 protein and/or glial fibrillary acidic protein (GFAP; Fig. 9.10 ). Rare cases may show divergent differentiation, including ganglion cells, melanin-containing cells, and rhabdomyoblasts; these component cells stain accordingly.

Olfactory neuroblastoma. A variety of neuroendocrine markers are positive: (A) Synaptophysin; (B) neuron-specific enolase; (C) CD56; (D) chromogranin.

Olfactory neuroblastoma. The sustentacular supporting framework cells are positive with S100 protein (A) or glial fibrillary acidic protein (B). The neoplastic cells are strongly reactive with calretinin (C).

The “small round blue cell” tumor differential is considered within this spectrum, and is discussed with each of the subsequent entities. Although lymphoma is not covered in this chapter, NK/T-cell lymphoma, 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. It is locally destructive, associated with necrosis, and must be separated from ONB and lymphoepithelial carcinoma. 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 ( Fig. 9.11 ). No neurofibrillary matrix or true rosettes are present. In some cases, rare, isolated areas of abrupt keratinization may be seen, suggesting NUT carcinoma.

Sinonasal undifferentiated carcinoma (SNUC). (A) SNUC showing a solid, lobular pattern of infiltration, with an area of central comedonecrosis. (B) Bone destruction is seen by cells with a high nuclear-to-cytoplasmic ratio, vesicular nuclear chromatin and a syncytial appearance. (C) CAM5.2 highlights the neoplastic cells. (D) Lymphovascular invasion is easily identified.

SNUCs are almost always strongly and diffusely positive for CK-pan, OSCAR, CK8, CAM5.2 ( Fig. 9.11 ), and p16 (see Tables 9.2 and 9.3 ), while positive with CK7, CK19, EMA, and p53 in about 50% of cases ( Fig. 9.11 ). p63 is positive in a few cases, but p40 and CK5/6 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 is negative. Curiously, most cases are strongly positive with p16 but lack any HPV DNA expression.

It may be difficult to separate SNUC from NPC, SCNEC, NUT carcinoma, and SMARCB1 -deficient carcinoma 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, p16, and p63. SCNEC must show histologic neuroendocrine differentiation, while tending to show a punctate or dot-like CK-pan and CAM5.2 reaction, neuroendocrine marker reactivity, and CD117 reaction. The recently recognized SMARCB1 (INI1)-deficient subset of poorly differentiated basaloid carcinomas shows lobules are tumors composed of a plasmacytoid to rhabdoid appearance of the neoplastic cells, lack keratinization, and may show necrosis. By definition, due to biallelic inactivation of SMARCB1 , there is a loss of SMARCB1 nuclear immunoexpression.

NUT Carcinoma

NUT carcinoma is an aggressive SCC subtype defined by NUTM1 gene rearrangements (see Tables 9.2 and 9.3 ). Patients are usually young, with a slight female predominance, presenting as 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. 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.

NUT carcinoma. (A) There is a sheet-like distribution of monotonous, primitive neoplastic cells that show an abrupt area of keratinization. (B) Strong, punctate granular nuclear reaction with NUT immunohistochemistry.

The neoplastic cells are immunoreactive with NUT ( Fig. 9.12 ), CK-pan, p63, p40, CK5/6, p16, and occasionally with neuroendocrine markers and CD34.

The differential diagnosis includes poorly differentiated SCC, SNUC, SMARCB1 -deficient carcinoma, Ewing sarcoma, 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. The cells will frequently have prominent, brightly eosinophilic nucleoli, intranuclear cytoplasmic inclusions, and opacified cytoplasm ( Fig. 9.14 ). Melanin is helpful when present. Mitoses are usually easily identified, as is tumor necrosis.

One of the most characteristic patterns of melanoma is a peritheliomatous growth (A). A slightly spindled to epithelioid growth of cells with mild pleomorphism is noted in this melanoma (B).

Melanoma has a variety of cell types. (A) Medium to large epithelioid loosely cohesive atypical melanocytes are associated with focal melanin pigment, seen below an intact respiratory epithelium. (B) A plasmacytoid pattern in this melanoma, showing prominent nucleoli and with several mitoses.

In general, S100 protein, SOX10, HMB-45, tyrosinase, Melan-A, MITF, and KBA are variably positive in MM, although S100 protein is the most sensitive and HMB-45 the most specific ( Figs. 9.15 and 9.16 ; see Table 9.3 ). 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. Overall, BRAF mutations are not significant in MM, although KIT and RAS mutations may be seen.

Mucosal melanoma is positive with several melanocytic markers, shown here highlighting a spindled cell melanoma. (A) S100 protein; (B) HMB-45; (C) tyrosinase; (D) Melan-A.

A mucosal melanoma with an epithelioid appearance, highlighted with vimentin (A) and KBA (B).

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 immunohistochemistry (IHC) panel can be ordered based on the anatomic site, age of the patient, pattern of growth, and further honed by the specific location of the immunohistochemical reaction. As an example, ONB shows a sustentacular S100 protein reaction that would not be seen in MM. A PNST would be positive with S100 protein and SOX10, but would not show HMB-45, Melan-A, or tyrosinase reactions ( Fig. 9.17 ). Mesenchymal chondrosarcoma also shows S100 protein reactivity, but is positive with SOX9, while negative with HMB-45, Melan-A, or tyrosinase ( Fig. 9.18 ).

A malignant peripheral nerve sheath tumor shows a spindle cell pattern with focal necrosis (A). The neoplastic cells in this case show a strong and diffuse S100 protein nuclear and cytoplasmic positive reaction, although usually not this strong (B).

A mesenchymal chondrosarcoma shows a “small round blue cell” appearance, set within a hemangiopericytoma-like vascular pattern (A). (B) Note the juxtaposition of the primitive cells to the mature cartilage. (C) The primitive cells have a slightly spindled appearance as they merge with the cartilage.

Sinonasal Neuroendocrine Carcinoma

Several tumors in the sinonasal tract have “neuroendocrine” differentiation (SNUC, ONB, pituitary adenoma, 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 neuroendocrine carcinoma are histologically similar to their lung counterparts (in this section, the small cell carcinoma type is considered). The tumors develop in middle-aged to older 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 ).

Sinonasal small cell neuroendocrine carcinoma is arranged in sheets, nests, ribbons and focally with pseudorosettes (A). The cells have a high nuclear-to-cytoplasmic ratio, showing delicate, fine nuclear chromatin distribution (B). Crush artifacts may belie the underlying neoplasm (C).

NECs, specifically small cell carcinoma, show a characteristic strong reaction for CK-pan and CAM5.2, usually in a punctate or dot-like pattern ( Fig. 9.20 ; see Tables 9.2 and 9.3 ). Cells are positive for p16 and EMA, focally with p63, rarely with calretinin, while negative with CK5/6, CK20, EBER, and FLI1 ( Fig. 9.21 ). The diagnosis is confirmed by staining with at least one neuroendocrine marker, including synaptophysin, chromogranin, CD56, CD57, NSE, and human achaete-scute homolog 1 ( Fig. 9.20 ). In some cases, the cells may be positive with S100 protein, CD117, and thyroid transcription factor 1 (TTF-1; Fig. 9.21 ). In rare cases, ectopic hormone or peptide expres­sion (calcitonin, adrenocorticotropic hormone, β-melanocyte stimulating hormone, serotonin, and parathyroid hormone) may be detected. There is usually a high Ki-67 proliferation index ( Fig. 9.21 ).

Neuroendocrine carcinoma (NEC) reacts with both epithelial and neuroendocrine markers. (A) CK-pan in a perinuclear dot-like pattern; (B) synaptophysin; (C) CD56; (D) chromogranin. Note the different staining patterns between antibodies: dot-like (Golgi), cytoplasmic, membranous, and focal.

Neuroendocrine carcinoma react with other nonneuroendocrine markers, including (A) p63; (B) thyroid transcription factor 1; (C) FLI1; (D) Ki-67 (>70% proliferation index).

Metastatic disease to the sinonasal tract from a pulmonary primary must always be considered, along with SNUC, Ewing sarcoma/primitive neuroectodermal tumor, pituitary adenoma, and NUT carcinoma. Pituitary adenoma does not have vascular invasion, lacks atypical mitoses, and will be reactive with various pituitary hormones or transcription factors. Ewing sarcoma usually shows CD99, ERG, and FLI1, while only showing weak to focal keratin and neuroendocrine reactions.

Pituitary Adenoma

Pituitary adenoma may develop in the sinonasal tract by direct extension from an intracranial tumor (most commonly in up to 3% of cranial primaries) or as an ectopic tumor that 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. 9.22 ), 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 ( Fig. 9.22 ), showing intranuclear cytoplasmic inclusions. The cells are supported in a vascularized to sclerotic stroma.

A pituitary adenoma identified below an intact respiratory epithelium. Note the syncytial appearance of the small cells with ample cytoplasm (A). Intranuclear inclusions and “invasion” around minor mucoserous glands may be seen (B). There is often limited tissue within a fibrovascular stroma (C). There is limited pleomorphism and inconspicuous mitoses (D).

Immunohistochemistry highlights the neuroendocrine nature, with synaptophysin (>95%), CD56 (>90%), NSE (>75%), chromogranin (>70%), and CD99 (~40%) 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. 9.23 and 9.24 ; see Table 9.3 ). Up to 60% of cases are positive for pituitary hormones: prolactin (most common), follicle stimulating hormone (FSH), luteinizing hormone, adrenocorticotropic horomone, thyroid stimulating hormone, and growth hormone, often plurihormonal ( Fig. 9.25 ). If required, pituitary transcription factors (such as Pit-1, T-pit, SF-1, ER-α, GATA-2) will help confirm the diagnosis in hormone silent cases. Calcitonin is rarely positive; CK7, CK5/6, and S100 protein are almost always negative.

A pituitary adenoma will be positive with CK-pan (A) and CAM5.2 (B) to a variable degree. Note the “dot-like” paranuclear staining, which is quite characteristic.

Pituitary adenoma reacts with different neuroendocrine markers: (A) Chromogranin (dot-like); (B) synaptophysin (membrane and cytoplasm); (C) CD56 (membrane); (D) neuron-specific enolase (granular cytoplasmic).

Pituitary adenoma reacts variably with prolactin: strong cytoplasmic reaction (A), strong but patchy (B). Other hormones may be positive, including follicle stimulating hormone (C) and adrenocorticotropic hormone (ACTH) (isolated cells) (D).

The differential diagnosis includes ONB, NEC, SNUC, paraganglioma, ES/PNET, and MM. The relative monotony of the cells, lack of lymphovascular invasion, no atypical mitoses, and tumor location help guide the immunohistochemistry evaluation (i.e., pituitary hormones would be performed after lymphoma, RMS, and 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, sinonasal tract, oropharynx and nasopharynx. Embryonal RMS tends to develop in the ear/mastoid, while alveolar RMS is more common in the sinonasal tract ( Figs. 9.26 and 9.27 ). Specifically, in the sinonasal tract, alveolar RMS develops in adults with a slight male predominance (1.2 : 1).

Rhabdomyosarcoma (RMS). (A) An embryonal RMS with a spindle cell population abutting cartilage. (B) Alveolar RMS showing an alveolar pattern with central necrosis. (C) Plasmacytoid to epithelioid cells, with eosinophilic cytoplasm. (D) Central area of dilapidation and loose alveolar architecture.

Rhabdomyosarcoma. The neoplastic cells react with a variety of markers, including (A) MYOD1; (B) myogenin; (C) desmin; (D) CD56.

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 ( Fig. 9.26 ). Focal tumor cell spindling ( Fig. 9.27 ) 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. 9.27, 9.28, and 9.29 ). 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, and up to 50% of cases will show a weak, focal, punctate, or dot-like CK-pan and/or CAM5.2 immunoreactivity ( Fig. 9.29 ). S100 protein and HMB-45 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 a break-apart FISH probe for the FOXO1 gene or by RT-PCR.

Alveolar rhabdomyosarcoma shows strong reactivity with: (A) vimentin; (B) desmin; (C) MYOD1; (D) myogenin.

Rhabdomyosarcoma also shows reactivity with other markers, including (A) SMA; (B) CD56; (C) CK-pan; (D) NSE.

Although the other “small round blue cell tumors” of the sinonasal tract are included in the differential diagnosis as presented in Table 9.3 , 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 an RMS ( Fig. 9.30 ). Malignant peripheral nerve sheath tumors (MPNST) may show rhabdomyoblastic differentiation, frequently referred to as a “Triton tumor” ( Fig. 9.31 ). Desmoplastic round cell tumor is a unique entity, which shows desmin, keratin, neuroendocrine marker, and Wilms tumor 1 (WT1) reactivity, but is negative with myogenin, MYOD1, and S100 protein ( Fig. 9.32 ); a FISH break apart 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.

Teratocarcinosarcoma shows carcinoma admixed with sarcoma (including rhabdomyosarcoma) and primitive elements in a teratoma-like pattern. (A) Rhabdomyosarcoma cells juxtaposed to epithelial islands. (B) Primitive cells showing focal rosette formation next to mucocytes. (C) Primitive cells adjacent to spindled sarcoma cells.

A triton tumor shows rhabdomyoblastic differentiation within a malignant peripheral nerve sheath tumor. Both S100 protein and muscle markers would be positive, although differentially expressed in each compartment. Desmin is shown in this case

(inset courtesy Dr. Roman Carlos).

A desmoplastic round cell tumor shows sclerosing fibrosis with primitive cells associated with areas of glandular differentiation (A). The neoplastic cells have a polyphenotypic expression with: (B) CK-pan (dot-like); (C) desmin (dot-like); (D) CD56 (cytoplasm and membrane).

Ewing Sarcoma/Peripheral Neuroectodermal Tumor

ES/PNET is a high-grade primitive small round tumor cell sarcoma with neuroectodermal differentiation and defined by the EWSR1 gene translocation, most common with FLI-1 . The Ewing family of neoplasms may be found in bone, soft tissue, and various parenchymal organs (e.g., 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. In all sites, there is a slight male predilection (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. 9.33 ), 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. 9.33 and 9.34 ). 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.

Ewing sarcoma show small to medium uniform cells (A) with a high nuclear-to-cytoplasmic ratio and delicate nuclear chromatin (B). Coagulative necrosis is frequently present (C).

Ewing sarcoma. (A) PAS without diastase highlights the glycogen; (B) FLI1 yields a strong nuclear reaction (endothelial nuclei serve as an internal control); (C) CD99 has a delicate membrane reaction, diagnostic for this tumor; (D) CK-pan is negative in this example.

ES/PNETs are strongly and diffusely reactive with CD99, usually in a membranous fashion ( Fig. 9.34 ); 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 NSE (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, either focally or diffusely, for cytokeratin CK-pan ( Fig. 9.34 ), CAM 5.2, and/or EMA, possibly related to expression of abnormal tight junctions. Rarely, desmin and chromogranin (2%) may be focally present, while WT1 and myogenin are negative.

The principal differential diagnoses include ONB, lymphoma, RMS, mesenchymal chondrosarcoma, small cell osteosarcoma, pituitary adenoma, 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 that 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 newly recognized BSS.

Sinonasal Intestinal-Type Adenocarcinoma

Adenocarcinomas of the sinonasal tract are separated into three major groups by the WHO: salivary gland type, intestinal-type adenocarcinomas (ITACs), and non-intestinal-type adenocarcinomas. Specifically, it is the intestinal-type and non-intestinal-type adenocarcinomas that are more frequently misinterpreted. 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 in women (4 : 1) and peaks in the fifth to seventh decades. In nonexposure 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 nonexposure-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. 9.35 ). In general, the colonic type has a tubuloglandular architecture, only rare papillae, nuclear pleomorphism, nuclear stratification, and increased 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.

Sinonasal intestinal-type adenocarcinomas are separated into a variety of different types based on pattern of growth and cellular components. (A) Colonic type with stratified nuclei and Paneth cells. (B) Papillary type looks identical to a tubular adenoma of the colon, including a muscularis mucosae. (C) Solid type shows stratified cells within a glandular architecture. (D) Mucinous type shows neoplastic cells suspended in lakes of mucin.

ITACs show both sinonasal and intestinal immunohistochemical reactions. ITACs will give a strong reaction for CK7 ( Fig. 9.36 ; Table 9.4 ), while also strongly positive for CK20, CDX2, SATB2, and MUC2, typically seen in intestinal-derived malignancies ( Fig. 9.36 ). Further, they will react with villin and MUC5, CEA-p ( Fig. 9.36 ), and a wide variety of neuroendocrine markers (synaptophysin, chromogranin, CD56) or even peptide hormones such as serotonin, gastrin, or somatostatin. p53 is usually overexpressed, although to a lesser degree in the mucinous type. Several other epithelial markers (EMA, B72.3) show variable reactivity. Tumors are negative with vimentin, actins, p63, and CK903. Interestingly, the low levels of KRAS, EGFR, and BRAF mutations in ITACs suggest a different pathogenesis, while also suggesting dif­ferences in targeted therapies.

Intestinal-type adenocarcinomas yield reactions that are similar to their intestinal counterparts, although also showing reactions unique to the sinonasal tract site. (A) CK20 (cytoplasmic); (B) CDX2 (nuclear); (C) CK7 (cytoplasmic); (D) CEA-p (luminal and cytoplasmic).

In general, without a good clinical history, imaging studies, or colonoscopy, it may be impossible to separate a metastatic tumor from the gastrointestinal 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, CDX2, 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 9.5 ), and serous adenoma. There is a recently recognized SNAC, referred to as “sinonasal renal-cell-like carcinoma,” a tumor showing monomorphous cuboidal to columnar glycogen-rich clear cells that lack mucin production. They histologically mimic metastatic renal cell carcinoma (RCC), while the renal cell-like carcinoma is showing CAIX and CD10 immunoreactivity, while negative with PAX8 and RCC marker ( Fig. 9.37 ).

(A) A metastatic clear cell renal cell carcinoma growing in an alveolar pattern, with clear cells easily identified (B). (C) A sinonasal renal-cell–like adenocarcinoma shows very similar histologic features, including a strong reaction with CAIX (D). PAX8 would help with the distinction.

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 (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. 9.38 ). 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 ( Fig. 9.38 ). Extravasated erythrocytes, mast cells, and eosinophils are noted throughout ( Fig. 9.38 ). Mitoses are inconspicuous. Tumor giant cells, lipomatous change, and hematopoiesis can be present, along with solitary fibrous tumor (SFT). Rare malignant cases are recognized.

Glomangiopericytoma show a patternless spindled to oval cell population below an intact respiratory epithelium (A). There is usually a very prominent to heavy perivascular hyalinization (B). Extravasated erythrocytes, eosinophils, and mast cells are usually easily seen (C).

The neoplastic cells are reactive with vimentin, actin (smooth muscle > muscle specific), β-catenin (nuclear; Fig. 9.39 ), 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. The tumor cells are negative with CD31, FVIIIRAg ( Fig. 9.39 ), keratin, SOX10, STAT6, TLE1, EMA, EBER, and CD117 (although the mast cells will be positive).

Glomangiopericytoma cells are highlighted by smooth muscle actin (A) and a strong and diffuse nuclear and cytoplasmic β-catenin reaction (B). There is no reaction with vascular markers such as CD31 (C) or FVIII-RAg (D).

The differential diagnosis includes LCH, SFT, BSS, synovial sarcoma, nasopharyngeal angiofibroma (NPAF), fibrosarcoma, PNST, desmoid fibromatosis, melanoma, fibrosarcoma, leiomyosarcoma, and meningioma ( Table 9.6 ). 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. A SFT may be concurrently present with glomangiopericytoma, but as a differential consideration, usually shows a lower cellularity, a higher deposition of wiry collagen ( Fig. 9.40 ), and a strong CD34, STAT6, and bcl-2 reaction ( Fig. 9.41 ). NPAF 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 tend to lack the rich vascularity of a glomangiopericytoma. Meningioma may be EMA and CK7 positive, but does not usually react with smooth muscle actin (SMA), muscle-specific actin (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, but a more diffuse and strong nuclear reaction with β-catenin.

(A–D) Solitary fibrous tumor shows a variety of different patterns as highlighted in this quartet of illustrations. There is a bland, spindled cell population set within a variably cellular collagenized stroma, often showing a vascular stroma.

Solitary fibrous tumor neoplastic cells show the following reactions: (A) Strong, nuclear STAT6; (B) CD34; (C) bcl-2; (D) CD99.

Biphenotypic Sinonasal Sarcoma

The newly recognized 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 nondescript, 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. 9.42 ). Rhabdomyoblastic differentiation may be seen, showing, interestingly, a different fusion partner by molecular analysis.

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