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Canine Digital Squamous Cell Carcinoma: Epidemiological, Histological and Immunohistochemical Study
50
ESVP/ECVP Proceedings 2012
148:1, 2013
CANINE DIGITAL SQUAMOUS CELL CARCINOMA: EPIDEMIOLOGICAL, HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY S. Belluco, E. Brisebard, D. Watrelot, T. Marchal and F. Ponce ICE, 2007-07-113, VetAgro-Sup, Marcy l’Etoile, France Introduction: Canine digital squamous cell carcinoma (CDSCC) is the most common tumour involving the digit of the dog. It is characterized by local invasiveness, possible multicentric development and a low metastatic potential. Dogs that develop multiple consecutive tumours or distant metastasis are often humanely destroyed. At present, the only factor used to predict outcome is histological grading. In oncology, the epithelialemesenchymal transition (EMT) is related to the invasiveness of tumour cells and to loss of epithelial marker expression and acquisition of a mesenchymal phenotype. This is the first large scale study to compare the tumour phenotype with prognosis in an attempt to discover prognostic markers. Materials and Methods: Samples from 49 dogs were analyzed histologically and immunohistochemically for the expression of pancytokeratin AE1/AE3, vimentin and E-cadherin. For each animal, epidemiological and clinical follow-up data were recorded. Results were correlated to the clinical outcome. Results: The Beauceron was among the three most overrepresented breeds; the forelimb was twice as likely to be affected as the hindlimb. The histological grade, the mitotic index and the expression of immunohistochemical markers were not correlated with the prognosis (local recurrence or metastasis). Conclusions: Neither the histological grading nor the EMT phenotype seem to be useful tools to define the prognosis for CDSCCs.
IMMUNOHISTOCHEMICAL DETECTION OF THE BCL-2 FAMILY MEMBERS BAK AND BAX IN CANINE LYMPHOMA M. Croci and F. Guscetti University of Zurich, Switzerland Introduction: The pro-apoptotic Bcl-2 family members Bak and Bax are essential for intrinsic apoptosis. Loss of expression of one or both of these proteins might contribute to tumourigenesis through impairment of apoptosis. In this study we validated antibodies for the immunohistochemical detection of these proteins in canine tissues and investigated their expression in samples of canine lymphoma. Materials and Methods: Commercially available antibodies against Bak and Bax were selected and validated based on their reactivity with canine recombinant proteins and with the endogenous proteins in UV-treated canine keratinocytes and non-neoplastic tissues using western blots and immunohistochemistry. The expression of these proteins was then assessed by immunohistochemistry in a series of previously classified lymphomas assembled in tissue arrays. Correlations of the expression of Bak and Bax with the immunophenotype and with previously investigated apoptosis-related markers were analyzed. Results: Immunolabelling of canine lymphomas showed a cytoplasmic signal for both proteins. The degree of immunoreactivity widely varied between tumours and was independent of the immunophenotype. Bax immunolabelling as assessed with two independent antibodies correlated positively with the number of active caspase-3-positive cells and negatively with immunolabelling for Bcl-x. Conclusions: Immunohistochemical data indicate that canine lymphomas express the pro-apoptotic proteins Bax and Bak variably. The association of Bax expression with apoptotic activity suggests a potential role of this protein with lymphomagenesis in dogs.
MACROPHAGE AND T CELL INFILTRATION IN FELINE ENDOMETRIAL ADENOCARCINOMAS M. Tavares Pereira *, R. Payan-Carreira *, A.L. Saraiva *,y and M.A. Pires* *CECAV-ECAV-UTAD, Vila Real and yEUVG, Coimbra, Portugal Introduction: Although considered rare tumours, feline endometrial adenocarcinomas (FEAs) seem to be more frequent than once thought. Aware of the importance of the immune system through tumour immunoediting, this study aimed to assess the infiltration of immune cells in FEA. Materials and Methods: Ten samples of papillary serous FEA (classified by examination of HE-stained sections) were used, together with six samples each of follicular and progestogenic stages of the oestrous cycle (controls). Immunolabelling was performed using antibodies against macrophages (MAC 387, Ab-Serotec, 1 in 100 dilution) and T cells (CD3, Dako, 1 in 50 dilution). Immune cells were counted in ‘hot spots’ within the tumour and in the peripheral stroma. Results: In tumours, T cells were predominant over macrophages, but both cell types were present in controls. A different distribution pattern was found in follicular and progestogenic stages; in the former T cells were abundant in the superficial layer, while in the latter they predominated in deeper endometrial layers. T cells and macrophages were significantly increased in tumours (P !0.05). An increased number of T cells were observed in the stroma surrounding the tumours, but macrophages were scarce in this location. Conclusions: Immune cells were significantly more numerous in papillary serous FEA than in the cyclic uterus. T cells predominated over macrophages in both intratumoural and surrounding tissues.
STANDARDIZATION OF COX-2 IMMUNOHISTOCHEMISTRY S. Belluco *, M. Castagnaro y, P. Carnier y, K. Chiers z, x na {, F. Queiroga k, I. Pires ±, S. Riffard ±, F. Millanta , L. Pe~ T. Scase # and G. Polton+ *VetAgro Sup, Marcy L’Etoile, France, yFaculty of Veterinary Medicine, Legnaro, Italy, zFaculty of Veterinary Medicine, University of Gent, Belgium, x School of Veterinary Medicine, Pisa, Italy, {Veterinary School, Complutense University Madrid, Spain, kUniversity of Tras-os-Montes and Alto Douro, Alto Douro, Portugal, Merial, Lyon, France, #Bridge Pathology Ltd., Bristol and +North Downs Specialist Referrals, Surrey, UK Introduction: Immunohistochemical expression of COX-2 in tumour cells is reported widely. Despite similarities between studies, results are difficult to compare. The aim of this study was to define a COX-2 immunolabelling technique that could achieve clinically plausible and comparable results in different pathology laboratories. Materials and Methods: Contiguous slides coming from 15 colorectal tumours, 15 oral squamous cell carcinomas and positive control slides from 1-day-old puppy kidney were labelled in eight European laboratories. Repeatability and reproducibility of labelling were evaluated and analyzed statistically. Results: Marked inter-laboratory and intra-laboratory variability was demonstrated using a standard technique. The technique was modified by inclusion of a new positive control tissue. Upper and lower limits of labelling intensity were defined. Repeatability was achieved, while reproducibility was not. Conclusions: The inclusion of positive control tissue with defined degrees of positivity allows repeatability of the labelling. Unfortunately, reproducibility remains a goal to be attained. Presently, this study indicates that comparison of results between different laboratories should not be made.
ESVP/ECVP Proceedings 2012
148:1, 2013
CANINE DIGITAL SQUAMOUS CELL CARCINOMA: EPIDEMIOLOGICAL, HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY S. Belluco, E. Brisebard, D. Watrelot, T. Marchal and F. Ponce ICE, 2007-07-113, VetAgro-Sup, Marcy l’Etoile, France Introduction: Canine digital squamous cell carcinoma (CDSCC) is the most common tumour involving the digit of the dog. It is characterized by local invasiveness, possible multicentric development and a low metastatic potential. Dogs that develop multiple consecutive tumours or distant metastasis are often humanely destroyed. At present, the only factor used to predict outcome is histological grading. In oncology, the epithelialemesenchymal transition (EMT) is related to the invasiveness of tumour cells and to loss of epithelial marker expression and acquisition of a mesenchymal phenotype. This is the first large scale study to compare the tumour phenotype with prognosis in an attempt to discover prognostic markers. Materials and Methods: Samples from 49 dogs were analyzed histologically and immunohistochemically for the expression of pancytokeratin AE1/AE3, vimentin and E-cadherin. For each animal, epidemiological and clinical follow-up data were recorded. Results were correlated to the clinical outcome. Results: The Beauceron was among the three most overrepresented breeds; the forelimb was twice as likely to be affected as the hindlimb. The histological grade, the mitotic index and the expression of immunohistochemical markers were not correlated with the prognosis (local recurrence or metastasis). Conclusions: Neither the histological grading nor the EMT phenotype seem to be useful tools to define the prognosis for CDSCCs.
IMMUNOHISTOCHEMICAL DETECTION OF THE BCL-2 FAMILY MEMBERS BAK AND BAX IN CANINE LYMPHOMA M. Croci and F. Guscetti University of Zurich, Switzerland Introduction: The pro-apoptotic Bcl-2 family members Bak and Bax are essential for intrinsic apoptosis. Loss of expression of one or both of these proteins might contribute to tumourigenesis through impairment of apoptosis. In this study we validated antibodies for the immunohistochemical detection of these proteins in canine tissues and investigated their expression in samples of canine lymphoma. Materials and Methods: Commercially available antibodies against Bak and Bax were selected and validated based on their reactivity with canine recombinant proteins and with the endogenous proteins in UV-treated canine keratinocytes and non-neoplastic tissues using western blots and immunohistochemistry. The expression of these proteins was then assessed by immunohistochemistry in a series of previously classified lymphomas assembled in tissue arrays. Correlations of the expression of Bak and Bax with the immunophenotype and with previously investigated apoptosis-related markers were analyzed. Results: Immunolabelling of canine lymphomas showed a cytoplasmic signal for both proteins. The degree of immunoreactivity widely varied between tumours and was independent of the immunophenotype. Bax immunolabelling as assessed with two independent antibodies correlated positively with the number of active caspase-3-positive cells and negatively with immunolabelling for Bcl-x. Conclusions: Immunohistochemical data indicate that canine lymphomas express the pro-apoptotic proteins Bax and Bak variably. The association of Bax expression with apoptotic activity suggests a potential role of this protein with lymphomagenesis in dogs.
MACROPHAGE AND T CELL INFILTRATION IN FELINE ENDOMETRIAL ADENOCARCINOMAS M. Tavares Pereira *, R. Payan-Carreira *, A.L. Saraiva *,y and M.A. Pires* *CECAV-ECAV-UTAD, Vila Real and yEUVG, Coimbra, Portugal Introduction: Although considered rare tumours, feline endometrial adenocarcinomas (FEAs) seem to be more frequent than once thought. Aware of the importance of the immune system through tumour immunoediting, this study aimed to assess the infiltration of immune cells in FEA. Materials and Methods: Ten samples of papillary serous FEA (classified by examination of HE-stained sections) were used, together with six samples each of follicular and progestogenic stages of the oestrous cycle (controls). Immunolabelling was performed using antibodies against macrophages (MAC 387, Ab-Serotec, 1 in 100 dilution) and T cells (CD3, Dako, 1 in 50 dilution). Immune cells were counted in ‘hot spots’ within the tumour and in the peripheral stroma. Results: In tumours, T cells were predominant over macrophages, but both cell types were present in controls. A different distribution pattern was found in follicular and progestogenic stages; in the former T cells were abundant in the superficial layer, while in the latter they predominated in deeper endometrial layers. T cells and macrophages were significantly increased in tumours (P !0.05). An increased number of T cells were observed in the stroma surrounding the tumours, but macrophages were scarce in this location. Conclusions: Immune cells were significantly more numerous in papillary serous FEA than in the cyclic uterus. T cells predominated over macrophages in both intratumoural and surrounding tissues.
STANDARDIZATION OF COX-2 IMMUNOHISTOCHEMISTRY S. Belluco *, M. Castagnaro y, P. Carnier y, K. Chiers z, x na {, F. Queiroga k, I. Pires ±, S. Riffard ±, F. Millanta , L. Pe~ T. Scase # and G. Polton+ *VetAgro Sup, Marcy L’Etoile, France, yFaculty of Veterinary Medicine, Legnaro, Italy, zFaculty of Veterinary Medicine, University of Gent, Belgium, x School of Veterinary Medicine, Pisa, Italy, {Veterinary School, Complutense University Madrid, Spain, kUniversity of Tras-os-Montes and Alto Douro, Alto Douro, Portugal, Merial, Lyon, France, #Bridge Pathology Ltd., Bristol and +North Downs Specialist Referrals, Surrey, UK Introduction: Immunohistochemical expression of COX-2 in tumour cells is reported widely. Despite similarities between studies, results are difficult to compare. The aim of this study was to define a COX-2 immunolabelling technique that could achieve clinically plausible and comparable results in different pathology laboratories. Materials and Methods: Contiguous slides coming from 15 colorectal tumours, 15 oral squamous cell carcinomas and positive control slides from 1-day-old puppy kidney were labelled in eight European laboratories. Repeatability and reproducibility of labelling were evaluated and analyzed statistically. Results: Marked inter-laboratory and intra-laboratory variability was demonstrated using a standard technique. The technique was modified by inclusion of a new positive control tissue. Upper and lower limits of labelling intensity were defined. Repeatability was achieved, while reproducibility was not. Conclusions: The inclusion of positive control tissue with defined degrees of positivity allows repeatability of the labelling. Unfortunately, reproducibility remains a goal to be attained. Presently, this study indicates that comparison of results between different laboratories should not be made.