MIB‐1 immunoreactivity correlates with biologic behaviour in canine cutaneous melanoma

The growth fraction of 68 canine cutaneous melanomas was determined by immunostaining with MIB‐1, a monoclonal antibody to a Ki‐67 epitope that recognizes all proliferating cells. Fifty tumours were classified histologically as benign and 18 as malignant. The Ki‐67 proliferative index (percentage of positive cells over 500 neoplastic cells) was low (< 15%) in 55 cases and high ( 15%) in 13 cases. High Ki‐67 proliferative index and histological malignancy were both associated with significantly poorer 2‐year survival (P < 0.0001). However, the predictive value of the Ki‐67 proliferative index (97%) was higher than the predictive value of classical histology (91%). The evaluation of the growth fraction by the Ki‐67 proliferative index is highly predictive of the biological behaviour of canine cutaneous melanoma.     

Flow cytometric evaluation of ki67 for the determination of malignancy grade in canine lymphoma

Ki67 is a nuclear antigen significantly correlated with degree of malignancy in human non‐Hodgkin lymphomas. We wanted to assess the ability of flow cytometric evaluation of Ki67 index (Ki67I) in differentiating the grade of malignancy in canine lymphomas. Ki67I was determined on lymph node aspirates of 90 immunophenotyped lymphomas classified according to the updated Kiel classification: 80 high grade (HG, 62 B cell and 18 T cell) and 10 low grade (LG, 3 B cell and 7 T cell) lymphomas. HG lymphomas showed significantly higher Ki67I compared with LG lymphomas (P < 0.0001). A significant difference in HG lymphomas was detected between B‐ and T‐immunophenotypes. Receiver operating characteristic (ROC) curve highlighted a high accuracy of Ki67I in recognizing HG lymphomas [area under the curve (AUC) = 99.4] and a cut‐off value of 12.2% was established (sensitivity = 96.3% and specificity = 100%). Thus, we suggest the combination of Ki67I flow cytometric determination and immunophenotype as a reliable tool to classify canine lymphomas.     

Argyrophilic nucleolar organizing regions and Ki67 equally reflect proliferation in fine needle aspirates of normal, hyperplastic, inflamed, and neoplastic canine lymph nodes (n = 101)

BACKGROUND: The count of argyrophilic nucleolar organizing regions (AgNOR) has been considered a useful variable that reflects cellular proliferation in canine lymph nodes, but it has not been compared with other markers of proliferation. Hypothesis: Ki67 and AgNORs are equally useful as markers of tissue proliferation in fine needle aspirates of canine lymph nodes. ANIMALS: A total of 101 dogs. MATERIAL AND METHODS: Prospective, observational study of a convenience sample of dogs. Two smears were prepared for a May-Gruenwald-Giemsa stain and a Ki67/AgNOR double stain. In addition, CD3/CD79a immunostaining was performed when cytologic examination revealed a lymphoma. The dogs were grouped as normal (n = 26), reactive hyperplasia (n = 25), lymphadenitis (n = 31), and lymphoma (n = 19), based on the physical examination and the cytologic findings. The AgNOR count/cell, AgNOR area/cell and the percentage of cells staining positive for Ki67 were evaluated in 100-167 cells (median, 113 cells) by using automatic image analysis. RESULTS: Mean (SD) AgNOR counts/cell were 1.36 +/- 0.19 in normal dogs, 1.55 +/- 0.26 in lymphadenitis, 1.65 +/- 0.32 in reactive hyperplasia, and 3.67 +/- 1.08 in lymphoma. The percentage of Ki67 positive cells was 2.67 +/- 0.99% in normal lymph nodes, 5.04 +/- 3.34% in lymphadenitis, 5.36 +/- 2.14% in reactive hyperplasia, and 30.2 +/- 10.8% in lymphoma. All variables were significantly higher in dogs with lymphoma compared with the other groups (P < .0001). The sensitivity and the specificity of the AgNOR count for diagnosing lymphoma were 95 and 96% at a cutoff value of >2.04 AgNORs/cell. The cutoff value for the Ki67 positive cells was >10.40% (sensitivity, 95%; specificity, 98%). CONCLUSION AND CLINICAL IMPORTANCE: The results indicated that both AgNOR and Ki67 counts were good diagnostic tools for assessment of proliferation in aspirates of canine lymph nodes.     

Cyclin D1 immunohistochemical expression and somatic mutations in canine oral melanoma

Canine oral melanoma (COM) is the most frequent tumour with oral localization in dogs. Copy number gains and amplifications of CCND1, a gene coding for Cyclin D1, are the most frequent chromosomal aberrations described in human non‐UV induced melanomas. Twenty‐eight cases of COM were retrieved from paraffin‐blocks archives. A total of 4 μm thick sections were immunostained with an antibody against human Cyclin D1 and Ki‐67. Cyclin D1 and Ki‐67 expressions were scored through two counting methods. DNA was extracted from 20 μm thick sections of formalin‐fixed paraffin‐embedded blocks. Pathological and surrounding healthy tissue was extracted independently. Cyclin D1 immunolabelling was detected in 69% (18/26) while Ki‐67 was present in 88.5% (23/26) of cases. Statistical analysis revealed correlation between two counting methods for Cyclin D1 (r = 0.54; P = .004) and Ki‐67 (r = 0.56; P = .003). The correlation found between Ki‐67 and Cyclin D1 indexes in 16/26 cases labelled by both antibodies (r = 0.7947; P = .0002) suggests a possible use of Cyclin D1 index as prognostic marker. Polymerase chain reaction analysis on CCND1 coding sequence revealed the presence of nine somatic mutations in seven samples producing synonymous, missense and stop codons. Since none of the single‐nucleotide polymorphisms was found to be recurrent, it is suggested that overexpression of Cyclin D1 may be the consequence of alterations of CCND1 upstream regions or other genetic aberrations not detectable with the methodology used in this study. Future studies are needed to verify the potential use of Cyclin D1 index as prognostic indicator and to highlight the molecular events responsible for Cyclin D1 overexpression in COMs.     

Immunocytochemical study of Ki-67 as a prognostic marker in canine mammary neoplasia

Background: Canine mammary tumors are challenging for clinicians and pathologists because of complex histologic classification, low specificity of cytologic diagnosis, and unpredictable biological behavior. In histologic specimens, expression of tumor proliferation marker Ki‐67, a nuclear nonhistone protein, has been shown to have prognostic value for canine mammary tumors and to correlate with malignancy and low survival rates. Objective: The objective of this study was to measure the proliferation index of canine mammary tumors by immunochemical detection of Ki‐67 in cytologic specimens and to determine its relationship to clinical and pathologic variables and patient outcome. Methods: Spontaneous mammary tumors from 31 female dogs were surgically excised. Imprint specimens for cytologic evaluation were wet‐fixed in ethanol; histologic specimens were prepared routinely. Immunostaining was performed with the PH 177 monoclonal antibody against Ki‐67; proliferation index was graded from negative to +++. Dogs were followed for 18 months. Multivariate logistic regression analysis was used to determine correlations between immunocytochemical results, tumor and clinical variables, and patient outcome. Results: Ki‐67 proliferation indices in cytologic specimens were significantly lower for nonmalignant tumors than for malignant tumors. High index values of Ki‐67 were positively correlated with metastasis, death from neoplasia, low disease‐free survival rates, and low overall survival rate. With the exception of 4 specimens for which cellularity was insufficient, positive expression of Ki‐67 in cytologic specimens correlated with that of histologic specimens. Conclusions: The prognostic value of the Ki‐67 index in canine mammary tumors by using wet‐fixed cytology imprint specimens was similar to that observed previously for histologic specimens. Immunocytochemical detection of Ki‐67 could improve the accuracy and value of cytology by providing safe and rapid information about malignancy and patient outcome.     

The histologic and epidemiologic bases for prognostic considerations in canine melanocytic neoplasia

The laboratory records from 384 dogs with a diagnosis of either melanoma or melanocytoma were selected for study. Significant negative determinants of patient survival for melanocytic tumors were: 1) metastasis, 2) mitotic index (MI), 3) nuclear atypia, 4) tumor score, 5) increasing size/volume, 6) the presence of deep inflammation, and/or 7) intralesional necrosis. In addition to these attributes, age was a significant determinant for tumors of the skin. For the feet and lips, 8) age and 9) junction activity negatively impacted survival. Mathematic models were constructed based on these significant determinants to predict the postsurgical outcome of melanocytic neoplasia. Melanocytic oral neoplasms comprised 19% (73/384) of the neoplasms; 92% of these were classified as malignant in the biopsy report, but malignant behavior (i.e., metastasis or recurrence) was observed in only 59% of cases. The prognostic model for oral tumors based on nuclear atypia provided the most accurate (89%) prediction of overall behavior. Melanocytic tumors of the feet and lips were also 19% (73/384) of the total population. Seventy-four percent were reported malignant, whereas only 38% actually demonstrated malignant behavior. The prognostic models based on both MI or nuclear atypia had an overall correct behavioral classification of 81%. Melanocytic tumors in the skin comprised 59% (227/384) of study specimens. Although 39% were reported as malignant, only 12% exhibited malignant behavior. A satisfactory predictive model that employed MI could not be constructed, but one using nuclear atypia gave an overall correct classification in 93.3% of the cases.     

Canine subcutaneous mast cell tumors: cellular proliferation and KIT expression as prognostic indices

Molecular assays are widely used to prognosticate canine cutaneous mast cell tumors (MCT). There is limited information about these prognostic assays used on MCT that arise in the subcutis. The aims of this study were to evaluate the utility of KIT immunohistochemical labeling pattern, c-KIT mutational status (presence of internal tandem duplications in exon 11), and proliferation markers--including mitotic index, Ki67, and argyrophilic nucleolar organizing regions (AgNOR)--as independent prognostic markers for local recurrence and/or metastasis in canine subcutaneous MCT. A case-control design was used to analyze 60 subcutaneous MCT from 60 dogs, consisting of 24 dogs with subsequent local recurrence and 12 dogs with metastasis, as compared to dogs matched by breed, age, and sex with subcutaneous MCT that did not experience these events. Mitotic index, Ki67, the combination of Ki67 and AgNOR, and KIT cellular localization pattern were significantly associated with local recurrence and metastasis, thereby demonstrating their prognostic value for subcutaneous MCT. No internal tandem duplication mutations were detected in exon 11 of c-KIT in any tumors. Because c-KIT mutations have been demonstrated in only 20 to 30% of cutaneous MCT and primarily in tumors of higher grade, the number of subcutaneous MCT analyzed in this study may be insufficient to draw conclusions on the role c-KIT mutations in these tumors.     

Prognostic significance of Ki67 evaluated by flow cytometry in dogs with high‐grade B‐cell lymphoma

Ki67 can discriminate between high‐ and low‐grade canine lymphomas, but its prognostic role in specific subtypes of the neoplasm is unknown. We assessed the prognostic significance of Ki67% (percentage of Ki67‐positive cells), evaluated by flow cytometry, in 40 dogs with high‐grade B‐cell lymphoma, treated with a modified Wisconsin–Madison protocol (UW‐25). The following variables were investigated for association with lymphoma specific survival (LSS) and relapse free interval (RFI): Ki67%, breed, sex, age, stage, substage, complete remission (CR). By multivariate analysis, Ki67% (P = 0.009) and achievement of CR (P = 0.001) were independent prognostic factors for LSS. Dogs with intermediate Ki67% (20.1–40%) presented longer LSS and RFI (median = 866 and 428 days, respectively) than dogs with low (median = 42 days, P < 0.001; median = 159 days, P = 0.014) or high (median = 173 days, P = 0.038; median = 100 days, P = 0.126) values. Determination of Ki67 is a prognostic tool that improves the clinical usefulness of flow cytometric analysis in canine high‐grade B‐cell lymphoma.     

Fascin‐1 expression in canine cutaneous and oral melanocytic tumours

Fascin‐1 expression was examined in 9 cutaneous melanocytomas and 47 oral melanomas. The cases were scored on the basis of extent and intensity of staining, and combined scores were calculated. Fascin‐1 expression was observed in 5/9 (56%) melanocytomas and 46/47 (98%) melanomas. The combined score for fascin‐1 was significantly greater in stage III/IV melanomas than in stage I/II melanomas (P < 0.05). In addition, strong fascin‐1 staining was associated with a significantly shortened survival time (P < 0.05). The results of this study suggest that fascin‐1 overexpression correlates with the malignancy of canine melanoma and has the potential to be a new immunohistochemical marker to predict the clinical course of canine melanoma. In addition, targeted therapy for fascin‐1 may represent a new strategy for the treatment of canine melanoma.     

Prognostic value of histologic stage and proliferative activity in canine malignant mammary tumors.

The aim of this study was to investigate the correlation between the histologic invasiveness (histologic stage) and various cell proliferation activity assays (quantity of argyrophil proteins associated with nucleolar organizer regions [AgNORs], mitotic activity, MIB1 [Ki67] immunohistochemical detection) for predicting the biologic behavior of malignant canine mammary tumors. Sixty specimens from malignant canine mammary tumors with no distant metastases (M0) at surgery were selected, and follow-up data were collected over a 2-year period. The histologic invasiveness was graded by histologic stage (stage 0 = tumors without stromal invasion; stage I = tumors with stromal invasion; stage II = tumors with neoplastic emboli in vessels), and the proliferative indices were expressed as MIB1 index (the percentage of nuclear area immunohistochemically stained by MIB1 antibody), mitotic index (the number of mitoses per 1,000 neoplastic cells), and AgNOR index (the ratio between mean AgNOR area of tumor cells and the mean AgNOR area of fibroblasts/lymphocytes). The measures of proliferative activity were compared among groups with different histologic stages, and the influence of different prognostic variables (histologic stage, AgNOR index, mitotic index, MIB1 index) on survival time was evaluated. A significant difference in the proliferation patterns was recorded between the different histologic stages for the mitotic index (P = 0.0006) and MIB1 index (0.0013). Among the different parameters considered, histologic stage (P < 0.05), AgNOR index (P = 0.0291), and MIB1 index (P = 0.014) revealed a significant association with prognosis in univariate analysis. AgNOR index for 1-year survival and histologic stage for 2-year survival were the most significant parameters influencing survival, as determined by multiple nonlinear logistic regression.     

Immunohistochemical characterization and evaluation of prognostic factors in canine oral melanomas with osteocartilaginous differentiation

Melanomas are the most common malignant oral neoplasm in dogs. Osteocartilaginous differentiation in oral melanomas is a rare feature described both in veterinary and human medicine. Here, 10 cases of this type of neoplasm were used to study their immunohistochemical, biological, and clinical characteristics. Reactivity for S100 and melan A antigen was evaluated, and 4 prognosis factors (mitotic index, invasiveness of epithelium, nuclear atypia, and proliferation index) were analyzed and correlated with the clinical course of the neoplasms after diagnosis. Immunohistochemical analysis of the studied neoplasms, including the osteocartilaginous areas, showed positive immunoreaction for S100 and melan A, except in one dog, which was negative for melan A. Analysis of the results showed that oral melamonas with osteocartilaginous differentiation have a clinical course similar to that of other melanomas in the oral cavity. Analysis of the mitotic index and the expression of proliferation marker Ki-67 could be useful tools for predicting the biological behavior of these neoplasms.     

Prognostic value of somatic focal amplifications on chromosome 30 in canine oral melanoma

Canine oral melanoma is the first malignancy of the oral cavity in dogs and is characterized by a local invasiveness and a high metastatic propensity. A better knowledge of genetic alterations is expected to improve management of this tumour. Copy number alterations are known characteristics of mucosal melanomas both in dogs and humans. The goal of this study was to explore the prognostic value of somatic focal amplifications on chromosomes (Canis Familiaris [CFA]) 10 and 30 in canine oral melanoma. The cohort included 73 dogs with oral melanoma confirmed by histology, removed surgically without adjuvant therapy and with a minimal follow‐up of 6 months. Epidemiological, clinical and histological data were collected and quantitative‐PCR were performed on formalin‐fixed paraffin‐embedded (FFPE) samples to identify specific focal amplifications. The 73 dogs included in the study had a median survival time of 220 days. Focal amplifications on CFA 10 and 30 were recurrent (49.3% and 50.7% of cases, respectively) and CFA 30 amplification was significantly associated with the amelanotic phenotype (P = .046) and high mitotic index (MI; P = .0039). CFA 30 amplification was also linked to poor prognosis (P = .0005). Other negative prognostic factors included gingiva location (P = .003), lymphadenomegaly (P = .026), tumour ulceration at diagnosis (P = .003), MI superior to 6 mitoses over 10 fields (P = .001) and amelanotic tumour (P = .029). In multivariate analyses using Cox proportional hazards regression, CFA 30 amplification (Hazard ratio [HR] = 2.08; P = .011), tumour location (HR = 2.20; P = .005) and histological pigmentation (HR = 1.87; P = .036) were significantly associated with shorter survival time. Focal amplification of CFA 30 is linked to an aggressive subset and constitutes a new prognostic factor.     

A matched observational study of canine survival with primary intraocular melanocytic neoplasia

A retrospective histopathologic study was performed to evaluate the effect of primary intraocular melanocytic neoplasia on canine survival. Tumor size, location within the globe, extent of infiltration, and mitotic index were analyzed for their potential to predict survival. A total of 244 cases of dogs with melanocytic tumors submitted to the Comparative Ocular Pathology Laboratory of Wisconsin from 1988 to 1998 were evaluated. Histopathologic criteria (mitotic index, cytologic features of anaplasia) were used to differentiate 188 benign melanocytomas from 56 malignant melanomas. Signalment evaluation of age, sex, and breed revealed similarities in both tumor populations, with the majority of tumors discovered in 9-year-old, female/spayed, mixed-breed dogs. A greater percentage of left eyes (66%) vs. right eyes (47%) was found in the melanoma population, but an equal distribution was found in the melanocytoma population (48% and 52%, respectively). The majority of tumors arose from the anterior uveal tract (79% in the melanocytoma and 95% in the malignant melanoma populations). The German Shepherd breed was predisposed in the limbal distribution. At the time of enucleation, most tumors had invaded the sclera, but did not show extrascleral extension (51% in the melanocytoma and 61% in the malignant melanoma populations). Survival analysis showed a significant difference in survival between control and malignant melanoma populations ( P  = 0.0081) and was suggestive of a difference between the melanocytoma and melanoma populations ( P  = 0.031). Tumor extension, tumor size, and mitotic index were not found to be reliable predictors of survival.     

Case of Bilateral Seminoma in a Trotter Stallion

This report describes a bilateral seminoma in a stallion. After slaughter, histological examination revealed that the tumour consisted predominantly of polyhedral tumour cells with large nuclei, obvious nucleoli and a small border of cytoplasm. The mitotic index was low and Ki67 staining revealed 4% nuclear staining. To our knowledge, this paper is the first using Ki67 staining as a method to evaluate the mitotic rate in a testicular seminoma in the stallion.     

Comparison of mitotic index and Ki67 index in the prognostication of canine cutaneous mast cell tumours

Proliferation markers are commonly used for prognostication of mast cell tumours. The aim of the study is to compare the relative abilities of Ki67 and mitotic index to predict survival in the same cohort of dogs with cutaneous MCTs. Histological grade, mitotic index and Ki67 index were performed in all samples and clinical information was obtained by a follow‐up questionnaire. Ninety‐five dogs were included in the study with a median follow‐up of 1145 days. Survival times varied significantly between categories of histological grade, mitotic index and Ki67 index. Multivariable analyses showed that the risk of dying due to MCT was similar in dogs with increased Ki67 index [hazard ratio, HR: 3.0 (95% CI 1.3–6.8)] or increased mitotic index [HR: 2.7 (95% CI 1.1–6.5)]. In conclusion, both mitotic index and Ki67 index were able to independently differentiate MCTs with worse prognosis. This distinction is particularly meaningful in selecting intermediate grade MCTs that may benefit from more aggressive local or systemic treatment.     

Canine limbal melanoma: 30 cases (1992-2004). Part 1. Signalment, clinical and histological features and pedigree analysis

OBJECTIVES: (1) To review the signalment, clinical, and histological features of canine limbal melanoma; (2) to perform pedigree analysis on breeds predisposed to limbal melanoma to establish if common ancestry exists; and (3) to investigate if any ancestral relationship exists between canine limbal melanoma and canine anterior uveal melanoma (CAUM). DESIGN: Retrospective study. ANIMALS STUDIED: Thirty dogs with limbal melanoma. METHODS: Medical records of patients were reviewed. Follow-up information was obtained by re-examination of patients or telecommunications with the referring veterinary surgeons or the owners. Pedigrees were analyzed for common ancestry amongst affected dogs. RESULTS The mean age (+/- SD) at diagnosis was 6.2 (+/- 2.75) years with a range from 1 to 11 years. There was a bimodal distribution of ages with a peak at 3-4 years and a peak at 7-10 years. There was no eye predilection or predisposition for sex or coat color. Twenty-five (83%) of the limbal melanomas occurred within a dorsal arc from the dorsomedial to the ventrolateral limbus. Golden retrievers were four times more common in the melanoma group compared to the Animal Health Trust population (P < 0.0001). Labrador retrievers were three times more common in the melanoma group (P=0.01). Pedigree analysis on eight Golden retrievers [limbal melanoma (n=5), CAUM (n=2) and diffuse ocular melanosis (n=1)], revealed a pattern of inter-relatedness consistent with the condition(s) being caused, at least in part, by a genetic mutation(s). A similar level of inter relatedness was evident in six Labrador retrievers (limbal melanoma (n=2) and CAUM (n=4)). In 5/22 cases (23%), histological features suggestive of malignancy were present including intratumor necrosis in 4/22 cases (18%) and cellular atypia in 1/22 cases (5%). CONCLUSIONS: In Golden and Labrador retrievers there is evidence that limbal melanomas, CAUM and ocular melanosis are at least in part heritable and that the same genetic mutation(s) may be causally associated with melanocytic disease at different ocular sites. The same genetic mutation(s) may be present in these two breeds. Histology should be performed on all cases to identify those with greater malignant potential.     

Association of argyrophilic nucleolar organizing regions, Ki-67, and proliferating cell nuclear antigen scores with histologic grade and survival in dogs with soft tissue sarcomas: 60 cases (1996-2002)

OBJECTIVE: To determine whether argyrophilic nucleolar organizing regions (AgNORs), Ki-67, and proliferating cell nuclear antigen (PCNA) scores were associated with histologic grade and survival in dogs with soft tissue sarcomas (STSs). DESIGN: Retrospective study. ANIMALS: 60 dogs with STSs. PROCEDURE: Medical records were examined and histologic specimens were reviewed. Tissue specimens obtained from archival materials were used to prepare sections for histologic staining for AgNOR and immunohistochemical staining for Ki-67 and PCNA labeling. Follow-up monitoring was obtained by reevaluation or telephone conversations with referring veterinarians or owners. RESULTS: 27 (45%) STSs were grade 1, 23 (38%) were grade 2, and 10 (17%) were grade 3. The mean and median AgNOR, Ki-67, and PCNA scores were determined, and significant positive associations among AgNOR and Ki-67 scores with histologic grade and mitotic score were detected. Fifty-four dogs had adequate follow-up examinations and were included in survival analysis and evaluation of prognostic factors. Overall median survival time was > 1,306 days. Twelve of 54 (22%) dogs died of tumor-related causes. Metastatic disease developed in 8 of 54 (15%) dogs. Results of univariate analysis indicated that increased mitotic score, increased AgNOR score, increased Ki-67 score, incomplete surgical margins, noncurative intent surgery, Ki-67 score greater than the median Ki-67 score, and AgNOR score greater than the median AgNOR score were prognostic factors for decreased survival time. Results of multivariate analysis indicated that increased AgNOR score was the only prognostic factor for decreased survival time. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that AgNORs and possibly Ki-67 should be routinely evaluated with histologic grading for STSs in dogs.     

Use of immunocytochemical techniques in canine melanoma

The staining patterns of the monoclonal antibodies S-100 and Melan-A in canine melanoma were assessed based on cytological specimens of six canine melanomas (four benign, two malignant). In addition, eight regional lymph nodes of the two dogs with malignant melanomas were stained using these markers. For reference, all specimens were also evaluated immunohistochemically using S-100 and Melan-A. To assess the immunocytochemical specificity of both antibodies, various canine tumours and normal tissues were stained. The immunocytochemical staining results of the canine melanomas and the regional lymph nodes showed high conformity with the immunohistochemical reactivity patterns for S-100 and Melan-A. The specificity of Melan-A was higher compared with S-100. Melan-A, in particular, may be helpful for the cytological diagnosis of canine melanoma.     

Relationship between NF-κB expression and malignancy of canine mammary gland tumor tissues

In this study, the nuclear expression of nuclear factor kappa B (NF-κB) in 48 tissues specimens from 25 canine spontaneous mammary gland tumor (MGT) patients was assessed by immunohistochemistry to compare their levels with clinical features, histological types, prognostic outcomes and proliferative activities, including the mitotic index (MI) and cylcinD1 expression. Twelve of eighteen (66.7%) malignant tumor tissues showed greater than 10% nuclear staining, while benign tumor and hyperplastic tissues showed less than 10% nuclear staining. Higher nuclear expression of NF-κB was positively correlated with larger tumor size, lymph node metastasis and higher MI; however, no correlation was observed with distant metastasis and cyclin D1 expression. Higher NF-κB nuclear expression correlated with shorter patient survival. These findings suggest that NF-κB is a useful prognostic factor for canine MGT patients.     

High COX‐2 expression in canine mast cell tumours is associated with proliferation,angiogenesis and decreased overall survival

COX‐2 overexpression is associated with several hallmarks of carcinogenesis such as proliferation, angiogenesis, invasion and metastasis. Fifty cases of canine mast cell tumours (MCT) were retrospectively evaluated and submitted to immunohistochemistry for COX‐2, CD31, Ki‐67, MAC‐387 and CD3. Furthermore its relationship with clinicopathological variables and overall survival (OS) was analysed. COX‐2 intensity (P = 0.016), but not COX‐2 extension nor score was associated with decreased OS and higher grades of malignancy according to Patnaik (P = 0.002) and Kiupel (P < 0.001) grading systems. Cox‐2 intensity was also associated with higher Ki‐67 scores (P = 0.009), higher mitotic index (P = 0.022) and higher microvascularization density (P = 0.045). No association was observed for COX‐2 intensity and CD3‐T lymphocyte (P = 0.377) and macrophage infiltration (P = 0.261) by MAC‐387 immunollabelling, suggesting an active role of COX‐2 in MCT oncogenesis mainly through proliferation and angiogenesis stimulation making it a potentially clinical relevant prognosis marker and therapeutic target.