Immunohistochemical Detection of Uroplakin III ...

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Immunohistochemical Detection of Uroplakin III, Cytokeratin 7, and Cytokeratin 20 in Canine Urothelial Tumors J. A. Ramos-Vara, M. A. Miller, M. Boucher, A. Roudabush and G. C. Johnson Vet Pathol 2003 40: 55 DOI: 10.1354/vp.40-1-55 The online version of this article can be found at: http://vet.sagepub.com/content/40/1/55

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Vet Pathol 40:55–62 (2003)

Immunohistochemical Detection of Uroplakin III, Cytokeratin 7, and Cytokeratin 20 in Canine Urothelial Tumors J. A. RAMOS-VARA, M. A. MILLER, M. BOUCHER, A. ROUDABUSH,

AND

G. C. JOHNSON

Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, MO Abstract. Immunohistochemistry for uroplakin III (UP III), cytokeratin 7 (CK 7), and cytokeratin 20 (CK 20) using commercially available antibodies was done in normal canine urinary bladder and 72 canine urinary bladder tumors that had been fixed in formalin and embedded in paraffin. Prolonged fixation (3–28 days) did not significantly alter the immunostaining for UP III. There was moderate reduction in the intensity for CK 7 and CK 20 after 1 week of fixation. UP III was detected in superficial (umbrella) cells and some intermediate cells of the normal urinary bladder, 7 of 7 transitional cell papillomas (TCPs), 50 of 55 transitional cell carcinomas (TCCs), and 4 of 5 metastatic TCCs. Staining was typically outlined in the plasma membrane, but diffuse or focal cytoplasmic staining was also observed. Intracytoplasmic lumina were usually positive for UP III. One squamous cell carcinoma of the bladder, 4 nonepithelial bladder tumors, and 285 nonurothelial tumors from different nonurinary locations were negative for UP III. CK 7 was detected in 7 of 7 TCPs, 53 of 54 TCCs, and 5 of 5 metastatic TCCs. The staining for CK 7 was diffuse cytoplasmic. CK 20 was detected in 1 of 7 TCPs, 37 of 54 TCCs, and 1 of 5 metastatic TCCs. The staining with CK 20 was cytoplasmic and weaker than with antibodies to UP III or CK 7. There was concurrent expression of UP III, CK 7, and CK 20 in 36 of 54 TCCs. UP III is a specific and sensitive marker for canine transitional epithelial (urothelial) neoplasms, detecting 91% of TCCs. Negative results may be observed with anaplastic tumors. Key words: plakin III.

Cytokeratin 7; cytokeratin 20; dogs; immunohistochemistry; neoplasia; urinary bladder; uro-

pancreatic carcinomas, transitional cell carcinomas, and endometrial carcinomas consistently expressed both cytokeratins.9 The mammalian urothelium consists of 6–10 layers of cells including superficial, intermediate, and basal cells.4,27 Superficial (also called umbrella) cells are characterized by a highly specialized plasma membrane that forms rigid-appearing plaques covering the apical surface of the urothelium.33 These plaques have a thick luminal leaflet and a thinner cytoplasmic leaflet that together form the asymmetric unit membrane (AUM). The cytoplasmic surface of these plaques appear to interact with a network of filaments suggesting that the plaques function to stabilize the apical surface during changes in volume of the urinary bladder.29 The AUM contains 16 nm in diameter hexagonal protein particles.32,33 The major protein components of the asymmetric unit are four transmembrane proteins named uroplakin (UP) Ia, Ib, II, and III.29,33 UPs are highly conserved through a broad range of mammalian species including human, monkey, sheep, pig, dog, rabbit, rat, and mouse.33 UPs are specific for transitional epithelium and its tumors.12,20,29 Immunohistochemical detection of UPs in domestic animals’ urothelial tumors has been reported only in cattle.3 The purpose of this study was to characterize the

Urinary bladder tumors account for 0.5–1.0% of all canine tumors and 2% of all malignant neoplasms.16 The majority of urinary bladder tumors in dogs are epithelial. Transitional cell carcinomas are the most common urinary bladder tumors in dogs and they have high metastatic potential.16 The immunohistochemical characterization of epithelial neoplasms of the urinary bladder has been done mainly with antibodies to cytokeratins (CKs). The transitional epithelium of the urinary tract (urothelium) contains simple-epithelium type cytokeratins (CKs 7, 8, 18, 19, 20) and stratified epithelium cytokeratins (CKs 13 and 17).18,19,30 The most common cytokeratins used in human pathology to distinguish urothelial tumors from other carcinomas are CKs 7 and 20;5,6,11,17 both cytokeratins are expressed in many urothelial carcinomas as well as in pancreatic carcinomas, cholangiocarcinomas, and ovarian mucinous carcinomas.5,7 Carcinomas from other origins (i.e., adrenal cortex, germ cell tumors, liver, lung, kidney, prostate, neuroendocrine, breast, mesothelium, uterus, and colon) usually have a different CK7/20 profile.6 There is a recent report of immunophenotyping of a variety of canine and feline tumors with CKs 7 and 20.9 Based on a few cases, none of the canine tumors examined had the CK 71/CK201 immunophenotype; several feline tumors, including 55

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Ramos-Vara, Miller, Boucher, Roudabush, and Johnson

Vet Pathol 40:1, 2003

Table 1. Reactivity of canine urinary tumors (n 5 72) with antibodies to uroplakin III (UP III), cytokeratin 7 (CK 7), and cytokeratin 20 (CK 20). Diagnosis

UP III*

CK 7*

CK 20*

Transitional cell papilloma Transitional cell carcinoma† Squamous cell carcinoma Lymphosarcoma Rhabdomyosarcoma Spindle cell sarcoma Metastatic transitional cell carcinoma

7/7 (100%) 50/55 (91%) 0/1 0/1 (0%) 0/2 (0%) 0/1 (0%) 4/5 (80%)

7/7 (100%) 53/54 (98.1%)‡ 0/1 ND ND ND 5/5 (100%)

1/7 (14.3%) 37/54 (68.5%)‡ 0/1 ND ND ND 1/4 (25%)‡

* Number of positive cases/total number tested. The number in parenthesis indicates percentage of positive cases. Criteria for interpretation of immunohistochemical results are included in materials and methods. † Two cases were in the ureter and renal pelvis. ‡ One case was not tested.

immunoreactivity of monoclonal antibodies (MoAb) to UP III, CK 7, and CK 20 in canine urinary bladder tumors fixed in formalin and embedded in paraffin. In addition, the specificity of MoAb PRO610608 to UP III for urothelial cells and their tumors was determined by immunostaining a variety of nonurothelial canine normal and neoplastic tissues. Materials and Methods Selection of cases

A total of 72 canine urinary bladder tumors was selected from the database of the University of Missouri—Veterinary Medical Diagnostic Laboratory. Tissues had been fixed in 10% neutral buffered formalin for an undetermined period of time and embedded in paraffin. Paraffin blocks had been stored from several days to more than 9 years. Hematoxylin and eosin (HE)-stained sections of these tumors were examined and tumors were classified by one author (JAR) following established criteria for urinary bladder neoplasms21 without knowledge of the previous diagnosis (Table 1). Those cases in which the morphologic diagnosis was not definitive were reviewed by two other pathologists (MAM, GCJ) to establish consensus. Tumors selected were 60 transitional cell carcinomas (TCCs), including 5 metastatic cases, 1 squamous cell carcinoma, 7 transitional cell papillomas (TCPs), 1 lymphosarcoma, 2 rhabdomyosarcomas, and 1 spindle cell sarcoma. When multiple samples of the same tumor were available, only one block was selected for immunohistochemical studies. To determine whether the grade of transitional cell carcinomas influenced the immunoreactivity for UP III, TCCs were graded from 1 to 3 (highest grade) according to a published system on the basis of the degree of cell differentiation; if different areas of the same tumor had different grades, the grade recorded was the highest one observed.31 To determine possible cross-reaction of MoAb to UP III, an additional 285 canine tumors or

lesions not associated with the urinary bladder were immunohistochemically stained with MoAb to UP III. Cases included 2 acanthomatous epulides, 11 adrenocortical carcinomas, 2 apocrine adenocarcinomas of the anal sac glands, 4 apocrine gland carcinomas, 8 basal cell tumors, 6 cholangiocellular carcinomas, 2 fibrosarcomas, 2 granular cell tumors, 9 granulosa cell tumors, 3 hair follicle tumors, 2 hemangiopericytomas, 4 hemangiosarcomas, 8 hepatocellular carcinomas, 5 histiocytomas, 13 interstitial cell tumors of the testis, 4 intestinal carcinomas, 10 lymphosarcomas, 2 malignant fibrous histiocytomas, 11 malignant peripheral nerve sheath tumors, 12 mammary tumors, 16 mast cell tumors, 11 melanomas, 4 mesotheliomas, 3 myxosarcomas, 1 nasal carcinoma, 6 osteosarcomas, 3 ovarian carcinomas, 18 pancreatic carcinomas, 7 pancreatic nodular hyperplasia, 13 perianal gland tumors, 2 pheochromocytomas, 8 plasmacytomas, 5 prostatitis, 9 pulmonary carcinomas, 10 renal carcinomas, 6 salivary carcinomas, 3 sarcomas of soft tissues, 13 sebaceous gland tumors, 5 seminomas, 5 Sertoli cell tumors of the testis, 4 smooth muscle tumors, 6 squamous cell carcinomas, and 4 thyroid carcinomas. A wide variety of normal tissues were also immunostained for UP III. Reagents and immunohistochemical procedure

All urinary bladder tumors were immunohistochemically stained for UP III (UP III), cytokeratin 7 (CK 7), and cytokeratin 20 (CK 20). The following primary antibodies were used: mouse MoAb to UP III (Research Diagnostics, PRO610608, clone AU1, diluted at 0.31 mg IgG/m1), mouse MoAb to CK 7 (Dako Corporation, M7018, clone OV-TL 12/30, diluted at 0.53 mg IgG/ml), and mouse MoAb to CK20 (Novocastra Laboratories, NCL-CK20, diluted at 0.75 mg IgG/ml). The antigen retrieval of choice for UP III, CK 7, and CK 20 was ready-to-use proteinase K incubated for 10 minutes at room temperature (RT);25 a parallel preliminary study using steam heat on citrate

Uroplakin III and Cytokeratins in Canine Urothelial Tumors


buffer, pH 6.0, incubated for 20 minutes at 90–95 C as an antigen retrieval produced inferior results in terms of reactivity or increased background when compared with proteinase K. Slides were put directly in Tris buffer after proteinase K treatment. The remaining incubations were also done at RT. The primary antibodies to CK 7 and CK 20 were incubated for 1 hour; incubation with MoAb to UP III was for 2 hours. For CK 7 and CK 20, a labeled streptavidin– biotin–peroxidase detection method (Dako Corporation, K0689) was used. For UP III, we used an avidin– biotin detection method initially, but there was intense nonspecific staining in some nonurothelial tumors (i.e., adrenal cortical tumors, sebaceous tumors, testicular interstitial cell tumors, mast cell tumors) that made interpretation difficult. Therefore, we decided to use the non–avidin–biotin system EnVision1/peroxidase (Dako Corporation, K4000), which did not produce diaminobenzidine (DAB) precipitate in the abovementioned tumors. Both detection methods followed manufacturer’s recommendations. Diaminobenzidine was used as chromagen. For negative controls, an unrelated monoclonal antibody of the same isotype and similar protein concentration was used instead of the primary monoclonal antibody. Additional antibodies were used in selected cases in which nonepithelial tumors were present, including muscle specific actin (DAKO, M0635), CD3 (DAKO, A0452), CD79a (DAKO, M7051), broad spectrum cytokeratins (DAKO, M0821), desmin (DAKO, M0760), and vimentin (DAKO, M7020) following standard protocols.25 Scoring of immunohistochemical results with MoAbs to UP III, CK 7, and CK 20

A semiquantitative (estimated) evaluation of the percentage of positive tumor cells was used: 1 (5–10% positive cells), 2 (11–50%), 3 (51–80%), and 4 (.80%). At least 5% of neoplastic cells were labeled to consider the tumor positive. A subjective grading system was used to determine the intensity of the reaction for these three antibodies in which 0 indicated no reaction, 1 weak reaction, 2 moderate reaction, and 3 intense reaction. Effect of prolonged fixation for detection of UP III, CK 7, and CK 20

Because the fixation time of most tumors was unknown, normal urinary bladder was fixed for the following days to determine the effects of prolonged fixation on immunohistochemistry: 2, 3, 5, 7, 11, 14, 21, and 28 days before processing. Results Cases selected

Sixty-five urinary bladder tumors were examined microscopically (Table 1) including 53 transitional cell

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carcinomas (TCCs), one squamous cell carcinoma, one lymphosarcoma, seven transitional cell papillomas (TCPs), two rhabdomyosarcomas, and one spindle cell sarcoma. In addition, two TCCs from the ureter and renal pelvis and five metastatic transitional cell carcinomas (two lymph node, one bone, one liver, one lung) were included in this study. The squamous cell carcinoma had fronds of epithelial cells forming clusters around keratin pearls. Typically, TCCs formed fronds or flat growths of epithelial cells that had variable amounts of vacuoles (also called intracytoplasmic lumina, acini, or microcysts).2,16,20 Sometimes, clusters of cells appeared to fuse their intracytoplasmic lumina. Invasion of the lamina propria-submucosa was a common feature of TCCs as well as invasion of the muscularis when it was available for examination. A scirrhous reaction accompanying infiltrating cells was also frequently seen. Glandular differentiation with acinar formation lined by a single layer of cuboidal cells was occasionally seen in TCCs. Tumors with a predominant glandular pattern were not present in this study. Only one transitional cell carcinoma was considered anaplastic. Nine (16%) of 55 TCCs had a histologic grade of 2; the other tumors (84%) were graded 3. Immunohistochemical results of MoAbs to UP III, CK 7, and CK 20 on normal urothelium

The UP III staining of the normal urothelium of the urinary bladder was very intense on the apical aspect of superficial (umbrella) cells but it was also strong in the cytoplasm of umbrella and upper layers of intermediate cells, sometimes with an unstained perinuclear halo. Basal cells did not react with antibody to UP III (Fig. 1). The transitional epithelium of the ureter and renal pelvis had similar immunoreactivity. In the normal urothelium, the staining with MoAb to CK 7 was usually diffusely cytoplasmic in umbrella and intermediate cells, with more intense staining of the apical cytoplasmic membrane of umbrella cells. For CK 20, the staining of the normal urothelium was weaker than with the other markers, diffusely cytoplasmic, and involving all layers of the epithelium except basal cells. The apical border of umbrella cells was more intensely labeled. Immunohistochemical results of MoAb to UP III on urinary bladder tumors

Fifty (91%) of 55 transitional cell carcinomas (TCCs) and 7 (100%) of 7 transitional cell papillomas (Fig. 2) were positive for UP III (Table 1). Thirty-two of 55 TCCs had .50% positive cells. In areas in which there was marked cell atypia but not invasion of the propria-submucosa (carcinoma in situ), the lowest layer of epithelial cells (interpreted as basal cells) was

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Fig. 1. Urinary bladder; dog. Normal urothelium. Fig. 1a. The staining for UP III is more intense in the apical border of the cytoplasmic membrane of umbrella cells but it is also present in the cytoplasm of superficial and intermediate cells. The lower layers of intermediate cells and basal cells are not stained (arrowheads). EnVision 1-peroxidase stain with Mayer’s hematoxylin counterstain. Bar 5 50 mm. Fig. 1b. Staining for CK 7 is more diffuse and is present in most umbrella and intermediate cells. Streptavidin–peroxidase stain with Mayer’s hematoxylin counterstain. Bar 5 50 mm. Fig. 1c. Staining for CK 20 is not present in basal cells (arrowheads). Streptavidin–peroxidase stain with Mayer’s hematoxylin counterstain. Bar 5 50 mm.



negative (Fig. 3). The staining with MoAb to UP III in TCCs was usually less intense than in adjacent areas of normal or hyperplastic epithelium (Fig. 4). It was uncommon to observe immunostaining of cells forming glands, and it was located in the plasma membrane (Fig. 5). Intravascular metastases were also reactive to UP III (Fig. 6). UP III labeling was frequently located in the plasma membrane and less commonly diffusely in the cytoplasm. The apical border of intracytoplasmic lumina was consistently and strongly labeled with this antibody. In rare occasions, the contents of intracytoplasmic lumina were strongly labeled (Fig. 7). The staining within the same tumor was heterogeneous in distribution and intensity. Staining was present in superficial cells and also in deeper layers of the growth (Fig. 4). The intensity of UP III labeling in the majority of urothelial tumors was strong. The only anaplastic transitional cell carcinoma examined was negative for UP III, but the adjacent normal urothelium was positive. The histologic grade of TCCs, based on most anaplastic nuclear appearance of a tumor,31 did

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not seem to correlate with the immunoreactivity for UP III. The squamous cell carcinoma was negative for UP III. Four (80%) of five metastatic transitional cell carcinomas had moderate to strong staining for UP III (Fig. 8). All nonepithelial urinary tumors were negative for UP III. The lymphosarcoma was positive for CD3 antigen and negative for CD79a antigen. Two (100%) of two rhabdomyosarcomas were positive for vimentin, desmin, and muscle actin, and negative for cytokeratins. The spindle cell sarcoma was positive for vimentin and negative for desmin and cytokeratins. Actin muscle immunostaining was not done in this case. Immunohistochemistry of MoAb to UP III on nonurothelial normal and neoplastic tissues

None of the nonurothelial normal tissues or nonurothelial tumors examined reacted with antibody to UP. Negative controls did not react with this antibody.

← Fig. 2. Urinary bladder; dog. Transitional cell papilloma. Numerous superficial urothelial cells covering the papillary projections (asterisks) are positive for UP III. EnVision 1-peroxidase stain for UP III with Mayer’s hematoxylin counterstain. Bar 5 300 mm. Inset: Detail of the staining. There is no staining of the lower layers of intermediate cells and basal cells. Bar 5 65 mm. Fig. 3. Urinary bladder; dog. Carcinoma in situ. There is marked atypia of the urothelium that contain numerous intracytoplasmic lumina. Note the lack of immunoreaction for UP III within nondysplastic epithelium under the carcinoma in situ (arrowheads) and adjacent hyperplastic epithelium (arrows). EnVision 1-peroxidase stain for UP III with Mayer’s hematoxylin counterstain. Bar 5 100 mm. Fig. 4. Urinary bladder; dog. Transitional cell carcinoma. Distinct staining for UP III of superficial and deeper neoplastic cells (asterisk). Note stronger staining of the adjacent nonneoplastic epithelium (arrowheads). EnVision 1-peroxidase stain for UP III with Mayer’s hematoxylin counterstain. Bar 5 300 mm. Fig. 5. Urinary bladder; dog. Transitional cell carcinoma with glandular differentiation. Note the membranous staining of glands (arrowheads). EnVision 1-peroxidase stain for UP III with Mayer’s hematoxylin counterstain. Bar 5 30 mm. Fig. 6. Urinary bladder; dog. Transitional cell carcinoma. Intravascular neoplastic cells. EnVision 1-peroxidase stain for UP III with Mayer’s hematoxylin counterstain. Bar 5 30 mm. Fig. 7. Urinary bladder; dog. Transitional cell carcinoma. There is heterogenous staining for UP III. Solid areas (arrows) have a diffuse cytoplasmic pattern. Cells with intracytoplasmic lumina have membranous staining (arrowheads). The contents of some lumina is also strongly positive. EnVision 1-peroxidase stain for UP III with Mayer’s hematoxylin counterstain. Bar 5 82 mm. Inset: Detail of intracytoplasmic lumina staining. Bar 5 38 mm. Fig. 8. Lymph node; dog. Metastatic transitional cell carcinoma. Neoplastic cells (asterisk) located in the subcapsular sinus have moderate staining for UP III. EnVision 1-peroxidase stain for UP III with Mayer’s hematoxylin counterstain. Bar 5 61 mm. Fig. 9. Lymph node; dog. Metastatic transitional cell carcinoma. Strong staining of neoplastic cells (asterisk) in subcapsular sinus for CK 7. Streptavidin–peroxidase stain for CK 7 with Mayer’s hematoxylin counterstain. Bar 5 82 mm. Fig. 10. Urinary bladder; dog. Transitional cell carcinoma. Streptavidin–peroxidase stain for CK 7 with Mayer’s hematoxylin counterstain. Bar 5 300 mm. Fig. 11. Urinary bladder; dog. Transitional cell carcinoma. The majority of neoplastic cells are strongly staining for CK 20. Note invasion of the propria-submucosa by neoplastic cells (arrowheads). Streptavidin–peroxidase stain for CK 20 with Mayer’s hematoxylin counterstain. Bar 5 122 mm. Fig. 12. Urinary bladder; dog. Transitional cell carcinoma. Fig. 12a. Distinct membranous staining includes intracytoplasmic lumina (asterisk) and is strong in the cytoplasm. Streptavidin–peroxidase stain for CK 7 with Mayer’s hematoxylin counterstain. Bar 5 30 mm. Fig. 12b. Similar field to 11 A. Weaker (both membranous and cytoplasmic) staining for CK 20. Streptavidin–peroxidase stain for CK 20 with Mayer’s hematoxylin counterstain. Bar 5 30 mm.

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Immunohistochemical results of MoAbs to CK 7 and CK 20 on urothelial neoplasms

Seven (100%) and one (14.3%) of seven TCPs were positive for CK 7 and CK 20, respectively. Fifty-three (98.1%) and 37 (68.5%) of 54 transitional cell carcinomas were positive for CK 7 and CK 20, respectively. The only TCC negative for CK 7 was the anaplastic type that was also negative for CK 20 and UP III. Two TCCs negative for UP III were positive for CK 20. Five (100%) of 5 and 1 (25%) of 4 metastatic TCCs were positive for CK 7 and CK 20, respectively (Fig. 9). The staining for CK 7 was in many cases heterogeneous with phenotypically similar cells not displaying immunoreactivity in the same section. The apical border of glandular structures and intracytoplasmic lumina was consistently and strongly positive (Fig. 10). With few exceptions, CK 7-positive cells were more numerous than UP III-positive cells in contiguous sections of the same tumor. The staining intensity for CK 7 was equal or stronger than that produced with MoAb to UP III. Staining for CK 20 was usually weaker than for UP III or CK 7 and sometimes difficult to distinguish from background although in some TCCs the intensity of the reaction for CK 20 was similar to or higher than that for CK 7 or UP III (Fig. 11). Some TCCs strongly positive for UP III and CK 7 were negative for CK 20. Concurrent (defined as the detection of several antigens in the same tumor but not necessarily in the same cell) expression of UP III, CK 7, and CK 20 was observed in 36 (66%) of primary TCCs and in one metastatic TCC. Coordinate expression of CK 7 and CK 20 was observed in 36 (67%) of primary TCCs (Fig. 12). Effect of prolonged fixation in the immunoreactivity of UP III, CK 7, and CK 20

Significant changes in UP III staining were not apparent at any given fixation time (2 through 28 days). For CK 7 there was a slight reduction in the intensity of the staining after 1 week of fixation, remaining stable through the rest of the period of examination, with more appreciable loss of reactivity on day 28. The loss of reactivity was more apparent in the lower layers of the urothelium. Staining for CK 20 was the least intense at any given time and was moderately reduced after day 11. Discussion This report describes the coordinate expression of UP III, CK 7, and CK 20 in canine normal and neoplastic urothelium fixed in formalin and embedded in paraffin. We found only two references of immunohistochemistry of UPs in domestic animals.3,10 A polyclonal antibody recognizing all four UPs labeled urothelial tumors of cattle with chronic enzootic hema-


turia.3 In the bovine normal urothelium, UPs were located in the cytoplasmic membrane of umbrella cells with no cytoplasmic expression.3 This staining pattern is similar to that found in other species,12,20,23,28 although weak staining of the cytoplasm of umbrella cells and intermediate cells has also been reported.12,20,23 In the only reported study of UPs immunostaining in dogs, the reaction was observed in all cell layers of the urothelium except the basal layer.10 In our case, the luminal aspect of umbrella cells had the strongest reaction, but we also observed strong cytoplasmic staining of umbrella cells and intermediate cells, with the lower layers of intermediate cells and basal cells consistently negative. The cytoplasmic staining is probably the result of localization of the antigen in fusiform vesicles of the cytoplasm.20,28 Fewer vesicles are present in intermediate cells.27 UPs are commonly detected in urothelial tumors of human, domestic, and laboratory animal species.3,12,15,20,23,24 Human TCCs are labeled with antibodies to UPs in about 53–88% of cases.12,20 In our study, the majority of TCCs (91% of primary tumors and 80% of metastatic tumors) and all transitional cell papillomas were positive for UP III. The pattern of staining was quite different to that observed in normal urothelium. Neoplastic cells usually exhibited plasma membrane staining without distinct polarity and less commonly diffuse cytoplasmic staining. The presence of staining in all sides of the cytoplasmic membrane may suggest that during neoplastic transformation, UPs may be redistributed.20 A distinct and strong staining of the rim of intracytoplasmic lumina was also very common in our series. UPs have also been detected in intracytoplasmic lumina of human TCCs.20 The origin of these lumina is controversial. Ultra structurally, some lumina lack the typical asymmetric unit membrane of urothelium but instead have glandular features,2 raising the possibility that UPs can be integrated into luminal membranes without the development of highly ordered AUM structures.20 Not all luminal structures in our series stained for UP III, which raises the possibility that not all lumina are structurally and functionally identical. Staining differences for UPs in luminal structures have also been reported in human TCCs.20 Interestingly, the contents of some intracytoplasmic lumina were strongly stained in some cases. The staining for UP III in some TCCs was multifocal with clusters of strongly positive cells and other areas in the same section containing nonreactive carcinoma cells, raising the possibility of false negative results if very small specimens or needle biopsies are obtained.12 The fact that areas of carcinoma in situ had distinct staining of umbrella and some intermediate cells but no staining of deeper layers of intermediate and basal cells contrasted with the diffuse and heterogeneous



staining of other areas in which invasion of the basement membrane was apparent. This pattern of staining might help to distinguish between carcinoma in situ and invasive transitional cell carcinomas. We did not find correlation between tumor grade of TCCs and detection of UP III. Interpretation of this result is difficult because the majority of tumors were of grade 3 according to a published grading system for canine TCCs.31 These results indicate that UP III can be expressed in high grade canine TCCs; however, the only truly anaplastic TCC (based not only on nuclear atypia but also on overall cell atypia and lack of features of urothelial epithelium) did not react with antibody to UP III. In humans, TCC grading system includes cell atypia and number of mitosis.8 High grade and metastatic human and bovine TCCs tend to be less reactive to UP III than low-grade TCCs.3,20 In another report, grade or tumor stage did not correlate with UP III reactivity.12 During experimental carcinogenesis, the ability of the urothelium to terminally differentiate is gradually lost as evidenced by the loss of UPs expression.23 Although uncommonly observed in our series, the staining of glandular structures with UP III in some TCCs supports the hypothesis that they originate from metaplastic differentiation from neoplastic urothelium.13,14 Up to 38% of TCCs have areas of squamous or glandular metaplasia.31 Final transformation of TCCs into squamous cell carcinoma or adenocarcinoma is a rare event in canine urinary bladder tumors in which an average of 3% are adenocarcinomas.16 In our series, we only observed one squamous cell carcinoma (negative for UP III) and no adenocarcinomas. We hypothesize that complete transformation of TCCs into adenocarcinomas or squamous cell carcinomas may occur concomitantly with the loss of expression of UP III. We did not observe UP III imunoreactivity in any of the nonurothelial normal or neoplastic tissues examined. The human literature on UPs reports similar results with some exceptions.20 Brenner tumors of the ovary are focally positive for UPs12,22 contributing to the belief that such tumors originate from transitional epithelium.34 Recently, UP Ib has been detected in corneal epithelium using frozen sections; however, there was no detection of UP Ia and UP III by reverse transcriptase-polymerase chain reaction.1 We did not observe staining for UP III in normal canine cornea. There was concurrent expression of UP III and CK 7, in 50 (96%) of 54 (one tumor was not examined for CK 7) TCCs. Cytokeratin 7 is immunohistochemically detected in 82–100% of human TCCs.5,7,18 Three of six cases of canine TCCs were positive for CK 7.9 In our series, there was poor correlation between immunoreactivity of UP III and CK 20 or CK 7 and CK 20. CK 7 was more sensitive than UP III for canine

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TCCs. The intensity of the reaction was also stronger for CK 7. But CK 7 is present in multiple human nonurothelial tumors (particularly in pulmonary carcinomas, ovarian carcinomas, salivary gland carcinomas, endometrial carcinomas, and thyroid carcinomas) and also in normal tissues and some canine tumors (i.e., glands, bile ducts, ovarian epithelium).7,9,26 CK 20 is also present in normal and neoplastic canine tissues.9 In summary, UP III is more specific than CK 7 and CK 20, although slightly less sensitive than CK 7 for the characterization of canine urothelial tumors. For diagnostic purposes, UP III is the marker of choice in canine urothelial neoplasms; CK 7 should be used in tumors negative for UP III but suspected of being TCCs. The use of antibodies to CK20 alone is not useful for diagnosis of urothelial tumors. Canine anaplastic TCCs may be negative for UP III, CK 7, and CK 20, but additional cases of anaplastic TCCs need to be examined to draw definitive conclusions. Prolonged fixation decreases the staining quality for CK 20 with minor loss of reactivity for CK 7 and no apparent loss for UP III. Acknowledgements We thank H. Wilson and D. Connor for photographic preparations.

References 1 Adachi W, Okubo K, Kinoshita S: Human uroplakin Ib in ocular surface epithelium. Invest Ophthalmol Vis Sci 41:2900–2905, 2000 2 Alroy J, Pauli BU, Hayden JE, Gould VE: Intracytoplasmic lumina in bladder carcinomas. Hum Pathol 10: 549–555, 1979 3 Ambrosio V, Borzacchiello G, Bruno F, Galati P, Roperto F: Uroplakin expression in the urothelial tumors of cows. Vet Pathol 38:657–660, 2001 4 Banks WJ: Epithelia. In: Applied Veterinary Histology, 3rd ed., pp. 48–67. Mosby Year Book, St. Louis, MO, 1993 5 Bassily NH, Vallorosi CJ, Akdas G, Montie JE, Rubin MA: Coordinate expression of cytokeratins 7 and 20 in prostate adenocarcinoma and bladder urothelial carcinoma. Am J Clin Pathol 113:383–388, 2000 6 Bostwick DG, Quian J, Ramnani DM: Prostate and bladder. In: Diagnostic Immunohistochemistry, ed. Dabbs DJ, pp. 407–458. Churchill Livingstone, New York, NY, 2002 7 Chu P, Wu E, Weiss LM: Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol 13:962–972, 2000 8 Eble JN, Young RH: Tumors of the urinary tract. In: Diagnostic Histopathology of Tumors, ed. Fletcher CDM, vol. 1, pp. 475–565. Churchill Livingstone, London, UK, 2000 9 Espinosa de los Monteros A, Fernańdez A, Millań MY, Rodrı´guez F, Herraéz P, Martıń de las Mulas J: Coordi-

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Request reprints from Dr. Jose´ A. Ramos-Vara, University of Missouri, Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, PO Box 6023, Columbia, MI 65205 (USA). E-mail: [email protected].