Original Article
DOI: 10.1111/vco.12095
Immunohistochemical expression of p63, Ki67 and 𝜷-catenin in canine transitional cell carcinoma and polypoid cystitis of the urinary bladder K. Hanazono1 , T. Nishimori1 , S. Fukumoto1 , Y. Kawamura2 , Y. Endo1 , T. Kadosawa1 and T. Uchide1 1
Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan 2 Department of Veterinary Pathology, School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
Abstract
Keywords canine, Ki67, p63, transitional cell carcinoma, 𝛽-catenin
Transitional cell carcinoma (TCC) is a urinary bladder tumour associated with high mortality in dogs. In this study, we investigated the feasibility of using p63, Ki67 or 𝛽-catenin as a clinical marker for predicting biological behaviour and prognosis in canine TCC. Expression levels of these proteins in TCC (n = 25), polypoid cystitis (n = 5) and normal urinary bladder (n = 5) were scored after immunohistochemical staining. The staining scores for p63 (P < 0.01) and 𝛽-catenin (P < 0.05) in TCC were significantly lower than those in normal urinary bladder and polypoid cystitis. In contrast, Ki67 (P < 0.01) staining scores in TCC were significantly higher than those in normal urinary bladder and polypoid cystitis. In TCC, low p63 expression was significantly related to the presence of vessel invasion (P < 0.05) and metastasis (P < 0.01) as well as short survival time (P < 0.05). These findings show that p63 could be a reliable marker for predicting prognosis in canine TCC.
Introduction
Correspondence address: T. Uchide, D.V.M., Ph.D. Department of Small Animal Clinical Sciences School of Veterinary Medicine Rakuno Gakuen University 582 Bunkyodai Midorimach Ebetu-shi Hokkaido 069-8501 Japan e-mail: [email protected]
Transitional cell carcinoma (TCC) is the most common tumour of canine urinary bladder, accounting for 50–75% of all reported tumours at this site.1,2 Because TCC generally is aggressive, with invasive and metastatic features, most affected dogs have advanced disease at the time of first presentation for clinical examination, with deep invasion into the urinary bladder wall and extensive metastasis to distant organs, including the lung, liver and bone.3,4 Therefore, appropriate clinical detection and diagnosis at an early stage is essential for obtaining a good outcome for treatment by minimum surgical excision and chemotherapy.3 Imaging diagnostic techniques including ultrasonography and computed tomography have been used for the early detection of urinary bladder
© 2014 John Wiley & Sons Ltd
tumours in veterinary medicine.3 However, when relying exclusively on imaging, it is difficult to distinguish TCC from other benign lesions including polypoid cystitis and benign bladder tumours.5 Definitive diagnosis of TCC has been based on cytological and pathological examination using cell and tissue samples obtained by catheter aspiration or biopsy via the urethra.6 Although these approaches are, at present, useful for diagnosis, it is difficult to predict clinical behaviour based solely on pathology. Molecular markers could support pathological examination of TCC tumours by providing additional information that can be used to predict their biological behaviour. In human medicine, a number of proteins have been recently reported as candidates for bladder cancer biomarkers.7 – 10 Among these, p63, Ki67
1
2 K. Hanazono et al.
and 𝛽-catenin have attracted attention because of accumulated evidence derived from basic and clinical studies supporting their clinical use.11 – 13 Although a few basic research studies in veterinary medicine have been reported, namely on TAG-72, uroplakin III, cytokeratin 7, COX-2, P-glycoprotein and survivin in TCC, the clinical importance of these proteins remains unclear.14 – 16 In this study, we investigated the feasibility of p63, Ki67 and 𝛽-catenin as clinical markers for predicting the biological behaviour and prognosis of canine TCC from a retrospective viewpoint.
Materials and methods
All experimental procedures in this study were carried out under approval (No.VH24A13) of the ethics committees of Rakuno Gakuen University.
Pathological examination Samples of urinary bladder tissues fixed in 10% neutral buffered formalin were embedded in paraffin wax and sectioned at 3 μm thickness. Tissues were stained with haematoxylin and eosin (HE) and examined histopathologically. TCC was diagnosed according to the World Health Organization Histological Classification of Tumours of Domestic Animals.17
Dogs
Immunohistochemistry
Five dogs with polypoid cystitis (2 males and 3 females) and 25 dogs with TCC (9 males and 16 females) were enrolled in this study on the basis of pathological records in the Laboratory of Veterinary Pathology at Rakuno Gakuen University. All polypoid cystitis and TCC tissues had been obtained via surgery. The mean ages of the polypoid cystitis and TCC cases at initial surgery were 9.6 ± 3.7 years (range, 4–13 years) and 9.7 ± 2.4 years (range, 7–13 years), respectively. There was no significant difference between the two groups. The canine breeds most often encountered were Shi-Tzu (n = 2, 40%) in polypoid cystitis cases and Shetland-sheepdogs (n = 5, 20%) in TCC cases. All TCC cases were subjected to partial (n = 11) or complete (n = 14) cystectomy. Post-operative chemotherapy used piroxicam alone (n = 6), mitoxantrone alone (n = 1), carboplatin alone (n = 1) or a combination of piroxicam and mitoxantrone (n = 3). No significant differences in survival time were observed between the two surgical methods or between the two groups with and without postoperative chemotherapy. TCC cases were followed from the time of operation until death. The mean follow-up period was 17 months (range, 1–60 months) for calculation of overall survival time. Five normal beagles (two males and three females) kept at our university were also enrolled to obtain normal urinary bladder tissue via necropsy. The mean age at necropsy was 7.4 ± 0.5 years (range, 7–8 years).
Serial sections were prepared from paraffinembedded tissues for immunohistochemical examination. Immunohistochemical staining was performed according to an avidin–biotin–peroxidase complex (ABC) procedure using a commercial kit (Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, CA, USA). Information on primary antibodies (against p63, Ki67 and 𝛽-catenin) including dilution, incubation time and pretreatment conditions for antigen retrieval is summarized in Table 1. Endogenous peroxidase activity was blocked by immersing tissues in 0.3% H2 O2 in methanol for 10 min. Staining was developed in 0.05% 3,3′ -diaminobenzidine solution. Negative control data were obtained by replacing the primary antibodies with phosphate-buffered saline (PBS). After immunostaining the proteins p63, Ki67 and 𝛽-catenin, scoring of the labelling was performed to estimate the degree of the protein expression in tumour tissues semi-quantitatively, based on previous reports.18 – 20 Scoring criteria are summarized in Table 2.
Statistical analysis Statistical difference among normal urinary bladder tissue, polypoid cystitis and TCC groups, or statistical independence of variables was assessed using the analysis of variance (ANOVA) or the chi square test, respectively. Survival curves were calculated by the Kaplan–Meier method and the difference among
© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12095
Markers for canine transitional cell carcinoma 3
Table 1. Primary antibodies and staining conditions
Clonarity
Isotype
Supplier
Dilution
Incubation time (h)
Antigen retrieval method
p63
Polycolonal H137
Rabbit IgG
1:100
16
Ki67
Monoclonal Mib-1
Mouse IgG
1:100
1
Polyclonal anti-serum
Rabbit IgG
Santa Cruz (Dallas, TX, USA) Dako (Glostrup, Denmark) Millipore (Billerica, MA, USA)
1:2500
16
Pressurized heating (121 ∘ C, 15 min) Pressurized heating (121 ∘ C, 15 min) Pressurized heating (121 ∘ C, 15 min)
Antibody
𝛽-catenin
Table 2. Scoring criteria for immunohistochemical labelling p63a
Score 0 1 2 3
No staining Focal stained or
DOI: 10.1111/vco.12095
Immunohistochemical expression of p63, Ki67 and 𝜷-catenin in canine transitional cell carcinoma and polypoid cystitis of the urinary bladder K. Hanazono1 , T. Nishimori1 , S. Fukumoto1 , Y. Kawamura2 , Y. Endo1 , T. Kadosawa1 and T. Uchide1 1
Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan 2 Department of Veterinary Pathology, School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
Abstract
Keywords canine, Ki67, p63, transitional cell carcinoma, 𝛽-catenin
Transitional cell carcinoma (TCC) is a urinary bladder tumour associated with high mortality in dogs. In this study, we investigated the feasibility of using p63, Ki67 or 𝛽-catenin as a clinical marker for predicting biological behaviour and prognosis in canine TCC. Expression levels of these proteins in TCC (n = 25), polypoid cystitis (n = 5) and normal urinary bladder (n = 5) were scored after immunohistochemical staining. The staining scores for p63 (P < 0.01) and 𝛽-catenin (P < 0.05) in TCC were significantly lower than those in normal urinary bladder and polypoid cystitis. In contrast, Ki67 (P < 0.01) staining scores in TCC were significantly higher than those in normal urinary bladder and polypoid cystitis. In TCC, low p63 expression was significantly related to the presence of vessel invasion (P < 0.05) and metastasis (P < 0.01) as well as short survival time (P < 0.05). These findings show that p63 could be a reliable marker for predicting prognosis in canine TCC.
Introduction
Correspondence address: T. Uchide, D.V.M., Ph.D. Department of Small Animal Clinical Sciences School of Veterinary Medicine Rakuno Gakuen University 582 Bunkyodai Midorimach Ebetu-shi Hokkaido 069-8501 Japan e-mail: [email protected]
Transitional cell carcinoma (TCC) is the most common tumour of canine urinary bladder, accounting for 50–75% of all reported tumours at this site.1,2 Because TCC generally is aggressive, with invasive and metastatic features, most affected dogs have advanced disease at the time of first presentation for clinical examination, with deep invasion into the urinary bladder wall and extensive metastasis to distant organs, including the lung, liver and bone.3,4 Therefore, appropriate clinical detection and diagnosis at an early stage is essential for obtaining a good outcome for treatment by minimum surgical excision and chemotherapy.3 Imaging diagnostic techniques including ultrasonography and computed tomography have been used for the early detection of urinary bladder
© 2014 John Wiley & Sons Ltd
tumours in veterinary medicine.3 However, when relying exclusively on imaging, it is difficult to distinguish TCC from other benign lesions including polypoid cystitis and benign bladder tumours.5 Definitive diagnosis of TCC has been based on cytological and pathological examination using cell and tissue samples obtained by catheter aspiration or biopsy via the urethra.6 Although these approaches are, at present, useful for diagnosis, it is difficult to predict clinical behaviour based solely on pathology. Molecular markers could support pathological examination of TCC tumours by providing additional information that can be used to predict their biological behaviour. In human medicine, a number of proteins have been recently reported as candidates for bladder cancer biomarkers.7 – 10 Among these, p63, Ki67
1
2 K. Hanazono et al.
and 𝛽-catenin have attracted attention because of accumulated evidence derived from basic and clinical studies supporting their clinical use.11 – 13 Although a few basic research studies in veterinary medicine have been reported, namely on TAG-72, uroplakin III, cytokeratin 7, COX-2, P-glycoprotein and survivin in TCC, the clinical importance of these proteins remains unclear.14 – 16 In this study, we investigated the feasibility of p63, Ki67 and 𝛽-catenin as clinical markers for predicting the biological behaviour and prognosis of canine TCC from a retrospective viewpoint.
Materials and methods
All experimental procedures in this study were carried out under approval (No.VH24A13) of the ethics committees of Rakuno Gakuen University.
Pathological examination Samples of urinary bladder tissues fixed in 10% neutral buffered formalin were embedded in paraffin wax and sectioned at 3 μm thickness. Tissues were stained with haematoxylin and eosin (HE) and examined histopathologically. TCC was diagnosed according to the World Health Organization Histological Classification of Tumours of Domestic Animals.17
Dogs
Immunohistochemistry
Five dogs with polypoid cystitis (2 males and 3 females) and 25 dogs with TCC (9 males and 16 females) were enrolled in this study on the basis of pathological records in the Laboratory of Veterinary Pathology at Rakuno Gakuen University. All polypoid cystitis and TCC tissues had been obtained via surgery. The mean ages of the polypoid cystitis and TCC cases at initial surgery were 9.6 ± 3.7 years (range, 4–13 years) and 9.7 ± 2.4 years (range, 7–13 years), respectively. There was no significant difference between the two groups. The canine breeds most often encountered were Shi-Tzu (n = 2, 40%) in polypoid cystitis cases and Shetland-sheepdogs (n = 5, 20%) in TCC cases. All TCC cases were subjected to partial (n = 11) or complete (n = 14) cystectomy. Post-operative chemotherapy used piroxicam alone (n = 6), mitoxantrone alone (n = 1), carboplatin alone (n = 1) or a combination of piroxicam and mitoxantrone (n = 3). No significant differences in survival time were observed between the two surgical methods or between the two groups with and without postoperative chemotherapy. TCC cases were followed from the time of operation until death. The mean follow-up period was 17 months (range, 1–60 months) for calculation of overall survival time. Five normal beagles (two males and three females) kept at our university were also enrolled to obtain normal urinary bladder tissue via necropsy. The mean age at necropsy was 7.4 ± 0.5 years (range, 7–8 years).
Serial sections were prepared from paraffinembedded tissues for immunohistochemical examination. Immunohistochemical staining was performed according to an avidin–biotin–peroxidase complex (ABC) procedure using a commercial kit (Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, CA, USA). Information on primary antibodies (against p63, Ki67 and 𝛽-catenin) including dilution, incubation time and pretreatment conditions for antigen retrieval is summarized in Table 1. Endogenous peroxidase activity was blocked by immersing tissues in 0.3% H2 O2 in methanol for 10 min. Staining was developed in 0.05% 3,3′ -diaminobenzidine solution. Negative control data were obtained by replacing the primary antibodies with phosphate-buffered saline (PBS). After immunostaining the proteins p63, Ki67 and 𝛽-catenin, scoring of the labelling was performed to estimate the degree of the protein expression in tumour tissues semi-quantitatively, based on previous reports.18 – 20 Scoring criteria are summarized in Table 2.
Statistical analysis Statistical difference among normal urinary bladder tissue, polypoid cystitis and TCC groups, or statistical independence of variables was assessed using the analysis of variance (ANOVA) or the chi square test, respectively. Survival curves were calculated by the Kaplan–Meier method and the difference among
© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12095
Markers for canine transitional cell carcinoma 3
Table 1. Primary antibodies and staining conditions
Clonarity
Isotype
Supplier
Dilution
Incubation time (h)
Antigen retrieval method
p63
Polycolonal H137
Rabbit IgG
1:100
16
Ki67
Monoclonal Mib-1
Mouse IgG
1:100
1
Polyclonal anti-serum
Rabbit IgG
Santa Cruz (Dallas, TX, USA) Dako (Glostrup, Denmark) Millipore (Billerica, MA, USA)
1:2500
16
Pressurized heating (121 ∘ C, 15 min) Pressurized heating (121 ∘ C, 15 min) Pressurized heating (121 ∘ C, 15 min)
Antibody
𝛽-catenin
Table 2. Scoring criteria for immunohistochemical labelling p63a
Score 0 1 2 3
No staining Focal stained or
