The pathological characteristics of enzootic nasal adenocarcinoma in goats

Five goats of the indigenous Chinese Anhui white breed, aged 0.8–2 years, from the same farm and commingled, became sick on a goat farm during the study period. The clinical manifestations of their disease included heavy respiratory sounds, unilateral or bilateral serous nasal discharge and reddening of the skin around the nostrils. Loss of appetite, emaciation, weaker rumination, depression and reluctance to stand were some of the clinical signs of the goats with more severe respiratory symptoms. Enzootic nasal tumour virus 2 (ENTV-2) was detected by PCR in the nasal secretion samples collected from five goats (9). The five diseased goats were dissected immediately after death. The nasal cavity was exposed by cutting the goat’s head along the median sagittal plane with a bone saw. Tumour samples were collected for pathological examination.

Tumour samples were fixed in 10% neutral buffered formalin solution and embedded in paraffin. The tissue sections were stained with HE and PAS and observed under an optical microscope.

Sections were then processed for immunohistochemical staining, which was achieved with anti-Pan-CK (1 : 500; Mouse Monoclonal Antibody TPB-0029; Typing Biotech Co., Nanjing, China), anti-CK7 (1 : 200; Rabbit Monoclonal Antibody DF7027; Affinity Biosciences, Cincinnati, OH, USA), anti-CKl8 (1 : 200; Rabbit Monoclonal Antibody AF0191; Affinity Biosciences), anti-OMP (1 : 500; Rabbit Monoclonal Antibody ab183947, abcam, Cambridge, UK) and anti-Kiel 67 (1 : 500; Rabbit Monoclonal Antibody TPB-0067; Typing Biotech Co.). Ethmoidal labyrinth samples from healthy Anhui white goats were used as controls and samples without the addition of primary antibody were used as negative controls. Immunohistochemical examination sections were routinely processed according to the antibody manufacturers’ instructions.

All sample sections were digitally scanned using a Pannoramic MIDI (3DHistech, Budapest, Hungary) and evaluated with Slide Viewer software v. 2.5 (3DHistech). Kiel 67-positive cells were counted using the NuclearQuant built-in image analysis application (3DHistech), which can quantify both the frequency and intensity of nuclear staining. Nuclei were appraised and given one of four different scores: 0 signified no staining, 1+ reflected low-intensity, 2+ moderate-intensity and 3+ high-intensity staining. All nuclei in the section were counted and given a graded score, and the number of nuclei rated as 3+ was divided by the total number of nuclei. The ratio of the Kiel 67-positive cells to tumour cell nuclei was found.

Tumour samples were also processed for routine TEM examination. The intranasal tumour was gently washed 2 or 3 times with sterile phosphate buffered saline (PBS, pH 7.2) to remove the mucus on the surface of the tumours. The tumour samples were cut to a size of 5 mm × 2 mm × 1 mm with a double-sided blade, fixed with 2.5% glutaraldehyde, dehydrated in graded concentrations of ethanol and embedded in epoxy resin. Thin sections cut with diamond knives were placed on copper grids and injected with uranyl acetate and lead citrate. The ultrathin sections were observed with a TEM (H-7650; Hitachi, Tokyo, Japan).

After incision and reflection of the frontal facial skin of the dead goats, the surface of the frontal bone was observed to be irregular with rounded foci of osteolysis (Fig. 1A). Removal of the frontal bone from the frontal sinus surface revealed that the frontal sinuses were filled with mucoid discharge (Fig. 1B). The five goats each had either unilateral (two) or bilateral (three) tumours in the nasal cavity. The tumours were pale white or pink and cauliflower-like, with a large amount of mucus covering the surface (Fig. 1C). The lesion was closely connected with the ethmoidal labyrinth and extended to the front of the nasal cavity along the turbinate bone (Fig. 1D). The ethmoid and turbinate bones showed varying degrees of destruction and displacement correlated with tumour size. The tumours had invaded the nasopharynx in the goats with severe clinical symptoms, which caused choanae obstruction (Fig. 1D). The affected portions of the ethmoid and turbinate bones were embedded in the tumour masses. No significant changes were observed in other tissues or organs.

Fig. 1.

Dissection pictures of the nasal cavity in Chinese Anhui white goats with enzootic nasal adenocarcinoma. A – varying degrees of osteolysis (white arrows) in the frontal bone; B – tumour surface covered with a large amount of mucus; C – pale white and cauliflower-like tumours with a granular surface (white arrows); D – goat’s head incised along the paramedian sagittal plane exposing a large tumour (white arrow) deep in the nasal cavity obstructing the nasopharynx

The caprine tumours were adenocarcinomas with predominantly well-differentiated and less frequently poorly differentiated regions. In histopathological sections, the tumour cells were organised into two structural patterns within the same adenocarcinoma: papillary formations in the superficial layer (Fig. 2A) and acinar formations in the deeper layer (Fig. 2B). The papillae were finger-like overall and were covered with a pseudostratified ciliated columnar epithelium. Tumour cells had large, rounded nuclei and thickened nuclear membranes. The nucleus was unevenly stained, pale and usually in the basal position (Fig. 2C). There was no significant change in the number and shape of goblet cells (Fig. 2C), which were interspersed among the epithelial cells. The stroma inside the papillary region was composed of connective tissues, including a large number of lymphocytes, macrophages, plasma cells and fibroblasts.

Fig. 2.

Representative haematoxylin-eosin and periodic acid–Schiff staining pictures of a caprine enzootic nasal adenocarcinoma. A – papillae located in the superficial layer of the tumour; B – acini located in the deeper layer of the tumour (broken black lines); C – papillae covered with a pseudostratified ciliated columnar epithelium; D – acinus consisting of a cuboidal epithelium of two to four layers (broken black line); E – remnant hyaline cartilage within the tumour tissue, with structure no longer intact (black arrows); F – epithelial-mesenchymal transition (black arrows) of tumour cells; G and H – mitotic figures (black arrow) of acinar and stromal tumour cells; I – a large number of PAS-positive granules on the cell surface; J – uneven, clumped purple granules aggregated in the acinar lumens (black arrows)

The internal tumour cells were arranged in acinar patterns. Most of the acinus consisted of a single layer of columnar or cuboidal epithelium, and the remainder was cuboidal epithelium of two to four layers (Fig. 2D). The borders of the acinar tumour cells were distinct, and the cytoplasm was eosinophilic or neutrophilic. The nuclei of acinar tumour cells were enlarged, round or oval, and located on one side of the cytoplasm or in the centre. The stroma between acini was loose connective tissue with arterioles and venules. There was no apparent tumour infiltration in the vessels and no apparent increase in lymphoid follicles. There was also remnant hyaline cartilage within the tumour tissue (Fig. 2E), probably ethmoid or turbinate bone. The normal bone structure was lost and its edge was crushed and eroded by the tumours.

Distinct epithelial–mesenchymal transition (EMT) was observed at the junction of acinar tumour cells and stromal cells in all affected goats. The epithelial cells began the morphological transition into elongated fibre cells (Fig. 2F). Mitotic figures of acinar (Fig. 2G) and papillary (Fig. 2H) tumour cells were rarely observed, one or none being noted per 10 high-power fields.

The PAS-positive granules were distributed in both the acini and papillae. There were more purple granules in pseudostratified ciliated columnar epithelia than there were on the papillae (Fig. 2I). No significant change was observed in the relative number or staining strength of goblet cells. The cytoplasm of the acinar cells was widely distributed with obvious and heterogeneous PAS-positive granules. Uneven, clump-like purple granules were aggregated in some acinar lumens (Fig. 2J).

Immunohistochemical examination of tumour tissues showed columnar epithelia on the superficial layer of the acini and papillae, and cuboidal epithelia deep in the acinar regions expressed CK18 and Pan-CK strongly throughout (Fig. 3A, B, G and H) and CK7 moderately throughout (Fig. 3D and E). Olfactory marker protein was not expressed in tumour epithelial cells (Fig. 3J and K). Kiel 67-positive nuclei were found in both tumour epithelial cells and stromal cells (Fig. 3M and N), resolving to a Kiel 67 index of approximately 23%.

Fig. 3.

Representative immunohistochemical pictures of enzootic nasal adenocarcinomatous and healthy goat ethmoidal labyrinth. A and B – strong, diffuse cytoplasmic expression of pancytokeratin (Pan-CK) in a tumour epithelium; C – positive cytoplasmic expression of Pan-CK in a normal ethmoidal labyrinth epithelium; D and E – moderate, diffuse cytoplasmic expression of cytokeratin 7 (CK7) in a tumour epithelium; F – positive cytoplasmic expression of CK7 in a normal ethmoidal labyrinth epithelium; G and H – strong, diffuse cytoplasmic expression of cytokeratin 18 (CK18) in a tumour epithelium; I – positive cytoplasmic expression of CK18 in a normal ethmoidal labyrinth epithelium; J and K – no expression of olfactory marker protein (OMP) in a tumour epithelium; L – positive cytoplasmic expression of OMP in a normal ethmoidal labyrinth epithelium; M and N – Kiel 67-positive nuclei in a tumour epithelium with a Kiel 67 index of approximately 23%; O – near absence of Kiel 67-positive nuclei in a normal ethmoidal labyrinth epithelium with a Kiel 67 index of close to 0%; P, Q and R – negative control for tumour tissue and a normal ethmoidal labyrinth. Black boxes (A, D, G, J and P) indicate the regions of higher magnification in the right-adjacent images (B, E, H, K and Q)

Ultrastructurally, retrovirus-like particles 120 nm in diameter were found both inside and outside the tumour cells surrounding the acinar lumen (Fig. 4A). The mature extracellular particles were spherical, surrounded by a lipid envelope, and had a round but eccentrically positioned inner core. Mature Betaretrovirus particles in the intracytoplasmic vacuole had a fine structure (Fig. 4A corner diagram). The immature intracytoplasmic particles had no envelope and were close to the cell membrane (Fig. 4B).

Fig. 4.

Ultrastructural manifestations of enzootic nasal adenocarcinoma. A – mature retrovirus-like particles in the intracytoplasmic vacuole and outside the cell (black arrows). Neighbouring tumour cells connected with tight junctions (white arrows). Scale bar = 2 μm. Corner view – details of the area indicated by the black box. Scale bar = 200 nm; B – immature intracytoplasmic particles (black arrowheads) and many secretory granules with heterogeneous density (white arrowheads) in the cytoplasm. Scale bar = 700 nm; C – sparse connections between stromal cells, and wide and obvious dilatation and vesiculation of the rough endoplasmic reticulum. Scale bar = 1 μm; D – small blood vessels in the stroma. Scale bar = 2 μm

The tumour cells were cuboidal in shape and were connected to each other by desmosomes and tight junctions (Fig. 4A). Short and sparse microvilli-like structures were observed on the cell surface. The nucleus had a round or oval shape and one or two nucleoli, with a predominance of euchromatin and without evident atypia (Fig. 4). There was an abundance of ribosomes and endoplasmic reticulum in the cytoplasm, and some rough endoplasmic reticula had wide and obvious dilatation and vesiculation (Fig. 4C). Moreover, in the cytoplasm near the acinar lumen, there were many secretory granules 200–700 nm in diameter. The secretory granules had an uneven electron density and a high density on one side, and resembled a bull’s eye in their shape (Fig. 4B). Small blood vessels and lymphocytes were also observed within the stroma (Fig. 4D).