CHAPTER 7 Oral Cavity, Gastrointestinal Tract, and Associated Structures

Claire B. Andreasen, Albert E. Jergens, Denny J. Meyer

Endoscopy has facilitated increased access to the mucosal surface of the gastrointestinal tract and enhanced the application of diagnostic cytology for its evaluation. It is an especially useful adjunct procedure when combined with the histologic examination of tissue for the complete assessment of gastrointestinal tract. The cytologic and histologic findings tend to be disparate when: (1) the specimens are obtained from different sites, (2) the lesion is deeply located in the lamina propria or submucosa precluding exfoliation, (3) surface-associated findings are lost during processing of the histologic sample, and (4) cell or tissue distortion (artifact) is present in either the cytologic or the histologic specimen.

In our experience, both touch imprint and brush cytologic preparations can provide useful information when concurrently examined with the histologic specimen (Jergens et al., 1998). Brush cytology tends to exfoliate more cells but also has the potential to induce hemorrhage and introduce leukocytes that may be mistaken for inflammation. Brush cytology often represents pathology of the deeper lamina propria compared to touch imprints, which reflect the surface and mucosal changes. The criteria that differentiate benignity and malignancy must be carefully evaluated when examining esophageal and gastrointestinal cytologic specimens since the cellular atypia associated with epithelial hyperplasia and regeneration as a consequence of inflammation can mimic epithelial neoplasia.

ORAL CAVITY

The most common reason for the cytologic examination of the oral cavity is for the evaluation of a mass or an ulcerative lesion. Radiographic findings will indicate if there is bone involvement.

Normal Cytology

Superficial and intermediate squamous epithelial cells are the commonly exfoliated cell type for the buccal cavity and the surface of the tongue (Fig. 7-1A). A variety of oropharyngeal bacteria can be seen in samples from the oral cavity. Noteworthy is Simonsiella sp. (Fig. 7-1B&C). Neutrophils are normally lost via transmigration through the mucous membranes into the oral cavity and may be observed in low numbers.

FIGURE 7-1 A, Oral cavity. Normal gingival epithelium. Dog. Keratinized stratified squamous epithelium forms papillary projections of the lamina propria (connective tissue). (H&E; LP.) B, Oral cavity. Normal cytologic findings. An angular squamous epithelial cell is covered with Simonsiella sp. (long arrow); an angular, partially keratinized squamous epithelial cell is located in the center, and the background is composed of numerous bacteria of varying size and shapes set in a lightly basophilic proteinaceous background. A stream of free nuclear protein from a ruptured cell is present (short arrow). (Wright; HP oil.) C, Oral cavity. Normal epithelium with flora. Dog. One intermediate squamous epithelial cell is covered with Simonsiella sp. (rounded rectangular structures with cross striations). A single erythrocyte is present in the lower right corner for size comparison. (Wright; HP oil.)

(A and C, Courtesy of Rose Raskin, Purdue University.)

Inflammation

Inflammatory diseases that can affect the oral cavity include immune-mediated diseases such as bullous disease, foreign bodies, dental disease, systemic manifestation of uremia, and bacterial, viral, and fungal infections (Guilford, 1996a). The inflammatory exudate can be composed of leukocytes, necrotic debris, and bacterial flora. Some raised plaque-like oral lesions with ulcerated surface will contain a pure eosinophilic population or mixed with eosinophils and neutrophils. Reports of an ulcerative eosinophilic stomatitis in the Cavalier King Charles spaniels (Fig. 7-2A&B) suggest this eosinophilic inflammatory response may occur concurrent with other affected systems and represent a hypersensitivity response in this breed (Joffe and Allen, 1995; German et al., 2002). In another condition, the undersides of the tongue may occasionally display a raised white mass which cytologically in addition to normal epithelium contains variable amounts of fine to coarse crystalline material. Most often it is extracellular but many crystals can be found within macrophages (Fig. 7-3A&B). This macrophagic or granulomatous (if giant cells are observed) inflammation is typical of a lingual calcinosis circumscripta. While urates may be considered, calcium is typical and can be confirmed with Von Kossa stain (Fig. 7-3C) or Alizarin red S (Marcos et al., 2006).

FIGURE 7-2 Same case A-B. A, Eosinophilic and neutrophilic stomatitis. Touch imprint. Dog. This nonpainful 2 × 2 cm raised plaque-like lesion with ulcerated surface appeared on the soft palate of a Cavalier King Charles spaniel. A mixed population of mostly nondegenerate neutrophils and eosinophils appear against a hemodiluted background. Both cell types were within normal reference ranges in circulation. (Wright; HP oil.) B, Ulcerative eosinophilic stomatitis. Histology. The ulcerated surface is densely infiltrated with a mixture of neutrophils and eosinophils. Adjacent to this area (not shown) was granulation tissue. However, granuloma formation was not observed. (H&E; IP.)

(A and B, Courtesy of Rose Raskin, Purdue University.)

FIGURE 7-3 Tongue. Calcinosis circumscripta. Touch imprint. Dog. Same case A-C. A, A white bump on the middle of this canine tongue was present for six months. Four highly vaculated and foamy macrophages appear against a refractile and granular background along with erythrocytes producing granulomatous inflammation. (Wright; HP oil.) B, Large coarse clear refractile granules are prominent in the background. (Wright; IP.) C, Same magnification as in B. Brown staining granules confirm calcium mineralization. (Von Kossa; IP.)

(A-C, Courtesy of Rose Raskin, Purdue University.)

Neoplasia

Epithelial, mesenchymal, and discrete (round) cell neoplasia can involve the oral cavity. As in other organ systems, the diagnostic sensitivity of cytology is highest for discrete cell neoplasia and lowest for mesenchymal tumors. This is due to reduced exfoliation and the difficulty differentiating neoplastic mesenchymal cells from reactive fibrocytes/fibroblasts that compose inflammatory/reparative lesions. Discrete cell neoplasia includes lymphoma, mast cell tumor, plasmacytoma, transmissible venereal tumor, and histiocytoma. The plasmacytoma can involve the oral mucous membranes, tongue, or mucosa of the digestive tract and be difficult to diagnose definitively without immunocytochemical or immunohistochemical characterization (Rakich et al., 1989). The most common epithelial neoplasm is squamous cell carcinoma (Fig. 7-4A&B). Less common is epithelial odontogenic neoplasia (Poulet et al., 1992) (Fig. 7-5A–C). Squamous cell carcinoma can occur anywhere in the oral cavity but in cats it often occurs in the frenulum of the tongue with early metastasis to the regional lymph nodes. An epulis is a common gingival mass of dogs or cats that can be a developmental, hyperplastic, inflammatory (Fig. 7-6A&B), or neoplastic lesion. A study of feline epulides (de Bruijn et al., 2007) determined the multinucleate cells of the giant cell epulis likely reflect an inflammatory response through osteoclast formation from mononuclear macrophages. These can be confusing lesions to evaluate cytologically because they can be composed of dental epithelial nests enrobed in stromal fibrous tissue. When the diagnosis is in doubt cytologically, it is prudent to pursue an excisional biopsy. Oral melanomas often are malignant and infiltrative, may contain abundant or only minimal pigment (amelanotic melanomas), and rapidly metastasize to the regional lymph nodes (Head et al., 2002) (Fig. 7-7A–C). Fibrosarcoma, chondrosarcoma, and osteosarcoma (Fig. 7-8) are the mesenchymal tumors that more commonly involve the oral cavity. Fibroma, hemangiosarcoma, and liposarcoma are less frequent (Bernreuter, 2008). Fibrosarcomas of the mandible and maxilla occasionally can have cytologic and histologic benign morphologic features but demonstrate aggressive biologic behavior (Ciekot et al., 1994). Neoplasia detected in the soft or hard palate may be extensions from the nasal cavity. In a study of lingual lesions (Dennis et al, 2006), about 4% of the cases in dogs involved the granular cell tumor, tumor of varied histogenesis (Head et al., 2002). Granular cell tumors (Fig. 7-9A–C) are mostly associated with the tongue than elsewhere in the oral cavity of older dogs and may occur in the cat as well. It is thought the majority of granular cell tumors arise from neuroectodermal origin and are set within reticulin fibers. Tumors appear as a sessile raised firm white mass which act benign. Cytologically, cells are individualized with eccentric nucleus with abundant eosinophilic or strongly periodic acid-Schiff-positive cytoplasmic phagolysosomal granules.

FIGURE 7-4 Oral squamous cell carcinoma. A, Touch imprint. This specimen is from a mass that extended from the frenulum of the tongue into the mandible of a cat. There are numerous squamous epithelial cells of varying immaturity with shapes that vary from oval to angular. Some of the cells show dyskeratosis (far left). Other abnormal morphologic features include variable nuclearto-cytoplasmic (N:C) ratios and variable staining. Numerous neutrophils are scattered amongst the neoplastic cells. Many show nuclear degeneration or are ruptured resulting in streaks of free nuclear protein. Squamous cells and keratin often induce neutrophilic inflammation. (Wright; HP oil.) B, Histology. Cords of pleomorphic squamous cells are separated by a fine fibrous stroma. Central cores of keratinocytes (arrows) demonstrate dyskeratosis (premature or abnormal keratinization of epithelial cells that have not reached the surface) and stain more intensively eosinophilic. (H&E; IP.)

FIGURE 7-5 A, Oral epithelial odontogenic tumor. Touch imprint. A cytologic impression of an epithelial cell neoplasm can be made based on the general morphology of the clustered interdigitating neoplastic epithelial cells. They show mild to moderate anisocytosis and anisokaryosis and the nuclei show mild to moderate shape variation. The lower group appears to be forming a disorganized acinar structure with a central lumen (arrow). The morphologic features are suggestive of an epithelial cell malignancy. The cytoplasm contains fine eosinophilic granules. Numerous erythrocytes surround the neoplastic cell clump. (Wright; HP oil.) B, Oral epithelial odontogenic malignancy. Histology. The dense neoplastic epithelial cell population shows mild central swirling with palisading cuboidal to columnar epithelial cells located at the periphery. Mitotic figures are present (arrows). The neoplastic cells are surrounded by a less-dense (pale pink) fibrovascular stroma. (H&E; IP.) C, Gingiva. Acanthomatous ameloblastoma. Histology. Dog. A layer of keratinizing epithelium (thin outer pink near size marker) covers the thick acanthomatous epithelium characteristic of this malignant neoplasm. This is an infiltrative tumor which forms cords of epithelium into the submucosa. (H&E; LP.)

(C, Courtesy of Rose Raskin, Purdue University.)

FIGURE 7-6 Gingiva. Giant cell epulis. Dog. Same case A-B. A, Aspirate. Aggregates of inflammatory cells composed of multinucleate histiocytes and individualized macrophages are prominent. Fibrovascular stroma creates a linear form (upper half) (Wright-Giemsa; IP.) B, Histology. Frequent anaplastic nuclear features are present in this solid mass of multinucleate cells with minimal stroma. (H&E; LP.)

(A and B, Courtesy of Rose Raskin, Purdue University.)

FIGURE 7-7 A, Oral malignant melanoma. Touch imprint. Pleomorphic polyhedral cells with variable N:C ratios, anisocytosis, and variable staining intensity are indicative of neoplasia. Abundant intracytoplasmic dark pigment (melanin) is noted in one cell (arrow). Most of the other cells do not contain obvious pigment (amelanotic) and can be easily confused with neoplastic epithelial cells or anaplastic mesenchymal cancer cells. (Wright; HP oil.) B, Buccal wall. Amelanotic melanoma. Imprint. Dog. Against a background of erythrocytes and bacteria is a sheet of anaplastic epithelium displaying variable and high N:C, coarse chromatin, multiple prominent nucleoli, anisokaryosis, and a mitotic figure (lower right end). Rare fine granules are present within the cytoplasm but granulation is not significant (Wright; HP oil.) C, Buccal wall. Amelanotic melanoma. Histology. Dog. Same case as in B. Poorly granulated individualized cells with pale open nuclei infiltrate the submucosa with some displaying junctional activity by their presence within the basal layer of the epithelium (arrows) support the diagnosis of melanoma. Hyperchromatic cells noted are neutrophils. (H&E; IP.)

(B and C, Courtesy of Rose Raskin, Purdue University.)

FIGURE 7-8 Oral osteosarcoma. Touch imprint. Pleomorphic mesenchymal cells show marked anisocytosis and anisokaryosis. The nuclei have stippled nuclear chromatin that contains multiple faintly stained variably sized nucleoli and are surrounded by moderately abundant basophilic cytoplasm with indistinct cell borders. The swirls of intercellular eosinophilic matrix produced by the neoplastic cells supports the cytologic impression of a malignant bone tumor. (Wright; HP oil.)

FIGURE 7-9 Tongue. Granular cell tumor. Histology. Cat. Same case A-C. A, Low magnification of a small raised lingual mass. (H&E; LP.) B, Dense network of stromal fibers separate the cells into small aggregates. (Reticulin; LP.) C, Stromal fibers create cords of individualized cells with abundant granular eosinophilic cytoplasm and hyperchromatic eccentric nucleus. (H&E; HP oil.)

(A-C, Courtesy of Rose Raskin, Purdue University.)

SALIVARY GLAND

Normal Cytology

Cytologic evaluation of salivary gland disease is diagnostically rewarding. The salivary gland also may be sampled accidentally when attempting to aspirate the submandibular lymph node. Cytologically, the salivary gland contains uniform secretory epithelial cells that are clustered and/or individual with eccentric, dark basophilic nuclei, and clear, vacuolated to foamy cytoplasm (Fig. 7-10). The cytoplasmic staining of the cells differs between serous cells (distinguishable) and mucous cells (may appear clear). Individual epithelial cells can be difficult to differentiate from macrophages, however, they have uniformly clear to finely vacuolated cytoplasm and do not contain phagocytic material. Eosinophilic-staining mucus is commonly observed and when present may cause erythrocytes to “stream” or line up in parallel rows (Fig. 7-11).

FIGURE 7-10 Salivary gland epithelial cells, normal. Touch imprint. The epithelial cells that compose this small cluster have abundant basophilic, granular-appearing cytoplasm that surrounds a dense, often eccentric nucleus. The morphologic features are consistent with normal secretory epithelium such as salivary gland. A large lymphocyte with a small rim of lightly basophilic cytoplasm (arrow) and a free round nucleus are located to the left of center. The surrounding erythrocytes can be used to judge the size of the cells. (Wright; HP oil.)

FIGURE 7-11 Sialocele (salivary mucocele). Aspirate. This specimen is an aspirate from a fluctuant submandibular swelling. The pinkish-blue mucus and occasional foamy macrophage along with a low number of neutrophils and erythrocytes are indicative of a sialocele. The rowing of erythrocytes (linear arrangement) suggests the presence of mucus. (Wright; HP oil.)

Hyperplasia

Hyperplasia is suspected when there is glandular enlargement and the epithelial cells appear relatively normal cytologically. The cells may be surrounded by abundant mucus. A differential consideration would be a sialocele.

Inflammation

The most common inflammatory lesion is caused by a salivary mucocele (sialocele) or ranula. A ranula is a cystic distension of an epithelial lined duct in the floor of the mouth. A sialocele is an accumulation of salivary secretions in nonepithelial lined cavities adjacent to the duct. While the cause is not known, trauma plus a developmental predisposition is proposed. The accumulated saliva stimulates an inflammatory reaction that changes over time. The initial inflammatory influx is composed of neutrophils and macrophages along with secretory epithelial cells set in an eosinophilic to basophilic mucus background (see Fig. 7-11). Lymphocytes replace the neutrophilic component and can become a prominent feature. Although the foamy macrophages can appear morphologically similar to the plump secretory epithelial cells, differentiation is not necessary when formulating the cytologic impression.

Neoplasia

The salivary adenocarcinoma is uncommon and demonstrates the general characteristics of epithelial malignancy that range from relatively well-differentiated to marked pleomorphism (Fig. 7-12A&B) (Spangler and Culbertson, 1991). The neoplastic epithelial cells can form acinar structures and some of the cells can have abundant retained cytoplasmic secretions that displace the nucleus to the periphery, forming a cell that appears similar to a signet ring. Mixed salivary neoplasms are rare and contain both neoplastic epithelial cell and mesenchymal cell components that can include bone and cartilage.

FIGURE 7-12 A, Parotid salivary gland. Adenocarcinoma. Aspirate. Cat. A cluster of immature cells display moderate anisokaryosis, coarse chromatin, prominent nucleolus and high N:C. Histopathology confirmed the malignant appearance. (Wright; HP oil.) B, Salivary gland. Adenocarcinoma. Histology. Salivary gland tissue from the tonsillar region demonstrates marked distortion of architecture (above pink connective tissue line) compared with the normal glandular structure (lower right) (H&E; IP.)

(A, Courtesy of Rick Alleman, University of Florida. B, Courtesy of Rose Raskin, Purdue University)

PANCREAS

Normal and Hyperplasia Cytology

The pancreas is not usually sampled unless a mass is detected. Cellular characteristics can rapidly deteriorate owing to extracellular pancreatic enzyme activity associated with pancreatitis. Hyperplastic nodules (see Fig. 2-4) can appear cytologically similar to normal epithelium. In these cases, the nuclear-to-cytoplasmic ratio is higher and nucleoli may be prominent.

Inflammation

Pancreatitis may cause focal abdominal fluid accumulations or effusions with the characteristics of either a modified transudate or a sterile purulent exudate (Fig. 7-13). If a proteinaceous background is present, it can have a “dirty,” moderately basophilic appearance that corresponds to saponified fat observed histologically. A hemorrhagic effusion is occasionally noted. Pancreatic cysts or abscesses can be detected as intra-abdominal masses and sampled by ultrasound-guided aspiration. The samples are often sterile and composed of neutrophils embedded in a proteinaceous background (Salisbury et al., 1988).