Usefulness of Ki-67 proliferation marker in the cytologic identification of liver tumors in dogs

BACKGROUND: Performing a biopsy is currently the best method of diagnosing liver disease. To reduce possible risk factors resulting from a biopsy, liver cytology can provide an alternative technique. The diagnostic accuracy of cytology for identifying liver tumors is, however, limited. The results of cytology might be improved by using immunochemistry for Ki-67, a proliferation marker, on liver cytology specimens. OBJECTIVES: The purpose of this study was to investigate the value of Ki-67 immunochemistry on liver cytologic specimens from dogs for identifying neoplastic diseases of the liver, by comparing the results to histologic findings. METHODS: Liver biopsy and cytology samples were obtained from 30 dogs with hepatic disease. All samples were evaluated by an anatomic pathologist and a cytopathologist. Parallel Ki-67 immunochemistry of histologic and cytologic samples was performed. The gradation of Ki-67 expression in histologic and cytologic samples was assessed. RESULTS: Cytologic specimens of liver tumors (n = 9) showed <50% Ki-67-positive cells. Twenty of 21 cases of non-neoplastic liver disease had no or few single Ki-67-positive cells. Using Ki-67, the diagnostic accuracy of cytologic evaluation was increased from 78% to 100% for malignant neoplasia. CONCLUSIONS: Based on the results of this study, the cytologic evaluation of liver together with Ki-67 immunochemistry can improve the diagnostic accuracy of cytology for liver neoplasia.     

Quantification of plasma DNA as a prognostic indicator in canine lymphoid neoplasia

Dogs have a similar incidence of spontaneous cancers as people, and a noninvasive test to monitor disease status in dogs would be of great value. Humans with cancer often have increased levels of cell‐free circulating DNA in their plasma, which has shown promise for diagnosis, prognosis and detection of residual disease. We hypothesized that dogs with cancer have increased circulating DNA compared with healthy dogs or dogs with non‐neoplastic diseases. Plasma DNA was measured in 40 healthy dogs, 20 dogs with non‐neoplastic diseases and 80 dogs with cancer. The reference interval for plasma DNA in healthy dogs was 1–15 ng mL^?1. Dogs with lymphoma and lymphoid leukaemia had significantly higher concentrations (range: 0–91 ng mL^?1, P < 0.0001). Antigen receptor rearrangement assays suggest that plasma DNA had the same clonality as the primary lymphoid tumours. Dogs with lymphoid neoplasia and plasma DNA >25 ng mL^?1 had shorter remission times than those with < 25 ng mL^?1 (P= 0.0116). In contrast to humans, where increased plasma DNA is seen in many diseases, dogs with nonlymphoid malignancies and non‐neoplastic diseases had plasma DNA concentrations similar to healthy dogs. This study shows that a portion of dogs with lymphoid neoplasia have increased tumour‐derived plasma DNA, which serves as a negative prognostic indicator.     

Immunocytochemical differentiation of canine mesenchymal tumors in cytologic imprint preparations

BACKGROUND: Immunocytochemical techniques are a potentially valuable diagnostic tool to support cytologic diagnosis in dogs. However, detailed studies of staining patterns and intensity in cytologic specimens of mesenchymal tumor types are lacking. OBJECTIVE: The aim of this study was to evaluate commercially available antibodies against human proteins for use in the characterization of canine tumors of mesenchymal origin in cytologic samples. METHODS: Immunocytochemical staining was performed on air-dried imprint specimens of biopsies obtained from 103 mesenchymal neoplasms and 14 metastatic lesions from 98 dogs. All specimens were stained with anti-cytokeratin AE1/AE3 and vimentin. Based on the histologic diagnosis, tumors of muscle, endothelial, histiocytic, and melanocytic origin also were stained with cell-specific antibodies. Staining intensity was subjectively graded and the percentage of positive tumor cells was estimated. RESULTS: All mesenchymal tumors and metastases, with the exception of mesotheliomas, were vimentin-positive and cytokeratin-negative; mesotheliomas (n=6) were positive for both vimentin and cytokeratin. Tumors of muscle (n=5), endothelial (n=15), and histiocytic (n=18) origin stained moderately to strongly positive in a majority of tumor cells with desmin, von Willebrand factor, and lysozyme, respectively. Malignant melanomas (n=15) had variable staining and a variable percentage of positive cells with Melan-A and S100. CONCLUSIONS: Our results indicate that immunocytochemical staining of canine cytologic specimens is a reliable and sensitive technique that may be of benefit for the differentiation of poorly differentiated mesenchymal tumors and metastases. Additional study is needed to assess the specificity of immunocytochemical stains in mesenchymal tumors.     

Expression of TopBP1 in canine mammary neoplasia in relation to histological type, Ki67, ERalpha and p53

TopBP1 is aberrantly expressed in human and feline mammary carcinomas, but expression of this BRCA1-related protein has not been investigated in canine mammary carcinomas. In this study, 132 canine mammary tumours (46 benign, 86 carcinomas) were examined immunohistochemically for expression of TopBP1, oestrogen receptor α (ERα), Ki67 and p53. Positive staining for TopBP1 was evident in all canine mammary lesions, although five samples had <20% positive cells. The number of samples with high levels of staining increased in different categories from benign mixed tumour to adenoma to carcinoma. Most TopBP1 staining was nuclear, but both nuclear and cytoplasmic staining were observed as the degree of malignancy increased, similar to human and feline mammary carcinomas. Benign mixed tumours, however, had more cytoplasmic staining than adenomas. Expression of p53 and the proliferation marker Ki67 increased from benign mixed tumour to adenoma to carcinoma, but the differences between benign and malignant tumours were more distinct than for TopBP1 expression. ERα expression decreased from malignant to benign tumours, although over half of the benign mixed tumours were negative. TopBP1 was expressed in canine mammary tumours at higher levels than has been reported previously for cats, although the shift in cellular localisation with malignancy was similar.     

MRI characteristics of canine prostatic neoplasia

Magnetic resonance imaging (MRI) has been used to evaluate dogs with suspected prostatic neoplasia, however, published studies describing MRI characteristics of canine prostatic neoplasia are currently lacking. The aims of the current retrospective case series study were to describe MRI findings of the pelvic region in dogs with a histopathologic or cytologic diagnosis of prostatic neoplasia. Retrospective analysis of these images was then performed by a board-certified veterinary radiologist for shared imaging characteristics. The most consistent characteristics were heterogeneous hyperintensity of the tumor on T2-weighted images (10/10) and short tau inversion recovery images (10/10), prostatic capsular margin distortion by the tumor (10/10), cavitations (10/10), complete effacement of the prostatic architecture (9/10), neurovascular bundle (NVB) compression or invasion (9/10), heterogeneous isointensity of the tumor on T1-weighted images (9/10), and strong contrast enhancement of the tumor (8/10). Additional features included an overlying pattern of distorted radiating striations (7/10), regional lymphadenomegaly (5/10), mineralization within the mass (5/10), urinary bladder trigone involvement (6/10), and post-prostatic urethral involvement (7/10). These findings supported the use of MRI as an adjunct imaging modality for diagnosis and therapeutic planning of prostatic neoplasia and including prostatic neoplasia as a likely differential diagnosis for dogs with these MRI characteristics.     

The relationship between tumour size and expression of prognostic markers in benign and malignant canine mammary tumours

Tumour size is considered one of the most important determinants of clinical staging in cancer patients. The aim of this study was to assess the value of tumour size as an indicator of the differentiation of mammary neoplasias in female dogs. The tumour, nodes metastates (TNM) system, based on primary lesion size, the extent of its dissemination to regional lymph nodes and the presence or absence of distant metastases, was applied to 120 female dogs diagnosed with mammary neoplasias. Paraffin blocks from 38 cases were selected and studied by immunohistochemical staining for prognostic and predictive markers of breast cancer. The Kaplan–Meier survival curve was estimated for 110 female dogs. Larger tumours (T3) were mostly malignant and showed lower expression of progesterone receptor and higher expression of cellular proliferation markers. Global survival time was shorter in female dogs with large tumour masses. This study highlights the importance of tumour size as a prognostic indicator of mammary neoplasias in female dogs.     

Interleukin-8 as a prognostic serum marker in canine mammary gland neoplasias

Mammary gland tumors in female dogs are an excellent model for the clinic-pathological, diagnostic and prognostic investigation of mammary neoplasias. Prognostic and predictive markers are effective in research and routine diagnosis. Interleukins play a fundamental role in cancer, with a particular function in tumor growth, invasion and metastatic potential. Interleukin-8 (IL-8) is known to possess tumorigenic and pro-angiogenic properties, and its overexpression is seen in a number of human tumors. IL-8 serum levels were determined and correlated with the clinic-pathological features and clinical evolution of mammary gland neoplasias in female dogs. IL-8 was measured by an immunoenzymatic assay in 30 female dogs with mammary neoplasias within a 12 month follow-up and in 50 control animals. The correlation between IL-8 concentration and clinical parameters was investigated. A statistically significant difference in the IL-8 serum levels was found in tumor-bearing dogs compared to the controls. In addition, when the individual parameters were evaluated, IL-8 content showed a positive correlation with the tumor progression, lymph node involvement, recurrence and death. Single and multivariate analyses showed associations between tumor recurrence, metastasis, high clinical staging and high IL-8, and also with the death risk. This was also consistent with the high IL-8 content in dogs showing tumor recurrence and metastasis. IL-8 superexpression has been detected in a number of human tumors, usually associated with a poor prognostic. Besides promoting angiogenesis, IL-8 is strongly related with the metastatic phenotype of mammary tumor cells. High IL-8 concentration was found in mammary gland cancer patients with advanced disease stages. Our results show that IL-8 can be used as a non-invasive prognostic marker for mammary gland cancer, and can be useful for the prediction of disease progression and recurrence in dogs with mammary neoplasias. The increased level of this cytokine acts as an independent prognostic marker of survival and the identification of animals with the poor prognostic.     

Ki-67 and Vascular Endothelial Growth Factor Expression in Intracranial Meningiomas in Dogs

Background: Tumor proliferation in human intracranial meningiomas can be defined by the reactivity of the monoclonal antibody MIB-1 to the Ki-67 antigen. Vascular endothelial growth factor (VEGF), a pro-angiogenic factor, is a predictive marker for survival of dogs with intracranial meningiomas.
Hypothesis: Ki-67 is expressed in canine intracranial meningiomas and is associated with VEGF expression. Ki-67 expression is a prognostic marker for patient outcome.
Animals: Seventy client-owned dogs with WHO grade I intracranial meningiomas.
Methods: Retrospective study assessing the degree of immunostaining for Ki-67 by MIB-1 and VEGF expression in intracranial meningioma tissue from dogs. MIB-1 Labeling Index (LI) was calculated with Image J NIH-software. Extent, intensity, and distribution of VEGF-expression was assessed semiquantitatively. Cross tabulations with Fisher's exact tests and nonparametric Spearman's rank correlations were performed to identify associations between VEGF expression and MIB-1 LI. Fifteen dogs underwent postsurgical radiotherapy and were included in survival analysis. The effect of MIB-1 LI on survival was examined by Kaplan-Meier and Cox proportional hazards regression procedures.
Results: Ki-67 staining was positive in 91% (64/70) and VEGF expression was detected in 96% (67/70). There was no significant association between VEGF expression and MIB-1 LI. MIB-1 LI was not associated with survival.
Conclusions and Clinical Importance: MIB-1 antibody can be used to document cell proliferation in intracranial meningiomas in dogs, but does not predict outcome. No association between VEGF as a marker of angiogenesis and tumor proliferation was found. Angiogenesis might be a more important predictor of meningioma activity in dogs than is Ki-67.     

Expression and significance of PTEN in canine mammary gland tumours

To explore the expression and clinical importance of the anti-oncogene phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in canine mammary gland tumours, PTEN expression was compared in 50 cases of canine mammary tumour and four examples of normal mammary tissue using real-time quantitative PCR. PTEN expression was similar in benign mammary tumours and normal mammary tissues (P>0.05), but was lower in malignant tumours than in normal mammary tissues or benign mammary tumours (P<0.001). PTEN expression was also low in the lymph node metastases of malignant mammary tumours. The expression profile of PTEN in malignant mammary tumours compared to those without lymph node metastasis varied significantly. Low-level PETN expression might play an important role in carcinogenesis and the progression of canine mammary tumours, and PTEN protein detection might be useful in evaluating tumour development and prognosis.     

Plasma free amino acid profiles of canine mammary gland tumors

The purpose of this study was to elucidate the relationship between plasma free amino acid (PFAA) levels and the clinical stages of mammary gland tumors (MGT) in dogs. PFAA levels in canines with malignant mammary tumors were decreased compared to those of healthy animals. The levels of aspartate and ornithine, in the dogs with tumor metastasis were significantly decreased when compared to those of dogs that did not have metastases. Results of this study indicate that PFAA levels could be a risk factor or biomarker for canine MGT metastasis.     

Prognostic value of regional lymph node status in canine mammary carcinomas

In this study, we have determined the prognostic value of the presence of the micrometastases and metastases greater than 2 mm in the regional lymph nodes for bitches with mammary carcinomas. The study involved 51 dogs diagnosed with a single malignant epithelial tumour in the 4th or 5th mammary gland. All animals underwent regional mastectomy; the 4th and 5th mammary glands were removed together with the inguinal lymph node. The lymph nodes were examined immunohistochemically using the anti-cytokeratin antibody, clone AE1/AE3. The bitches were followed up every 6 months for 2 years after surgery to determine the disease-free survival (DFS) and overall survival (OS). The Kaplan-Meier analysis showed a statistically significant difference in DFS and OS only between the group of bitches without metastases and the group with lymph node metastases greater than 2 mm. No significant differences between these two groups versus bitches with lymph node micrometastases were found.     

Serum alkaline phosphatase isoenzyme activities in canine malignant mammary neoplasms with and without osseous transformation

BACKGROUND: Increased serum activity of total alkaline phosphatase (TALP) has been found in dogs with mammary neoplasms, especially malignant mixed tumors. We hypothesized that the bone isoenzyme of alkaline phosphatase (BALP), a specific indicator of osteoblastic activity and bone formation, may contribute to increased TALP in dogs with mammary neoplasms with osseous transformation. OBJECTIVE: The purpose of this study was to compare serum TALP, BALP, and other ALP isoenzyme activities in dogs with mammary malignant neoplasms with and without osseous transformation. METHODS: Twenty-one female dogs with malignant mammary neoplasms were compared with 21 clinically healthy, age-matched female control dogs. Physical, clinicopathologic (including preprandial and postprandial serum bile acids, ACTH stimulation, and low-dose dexamethasone suppression tests), radiographic, and ultrasonographic examinations were performed on all dogs with tumors to assess coexisting conditions. On the basis of histologic examination of excised tumors, dogs were further classified as having epithelial (n = 11) or mesenchymal/mixed (epithelial-mesenchymal) (n = 10) neoplasms, the latter of which had histologic and radiologic evidence of bone formation. Serum TALP, BALP, liver alkaline phosphatase (LALP), and corticosteroid-induced alkaline phosphatase (CALP) activities were measured using biochemical methods. RESULTS: Dogs with malignant mammary tumors had significantly higher (P < .05) median serum TALP (170 U/L), BALP (59 U/L), LALP (49 U/L), and CALP (24 U/L) activities, compared with control dogs (81, 32, 37, and 5 U/L, respectively). Significantly higher activities of BALP and LALP were found in dogs with epithelial neoplasms; whereas, only CALP activity was higher in dogs with mesenchymal/mixed neoplasms. There was no significant difference in TALP or isoenzyme activitities between epithelial and mesenchymal/mixed groups. CONCLUSION: BALP activity is increased in some dogs with malignant mammary tumors but does not account for the increase in TALP in dogs with neoplasms that have osseous transformation.     

Identification of genetic variation in 11 candidate genes of canine mammary tumour

The incidence of canine mammary tumours (CMTs) differs significantly between breeds, strongly supporting an influence of genetic risk factors. We aimed at identifying germline genetic variations in mammary tumour-associated genes in dogs and survey whether these might alter the encoded proteins. We sequenced 11 genes (BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EGFR, ESR1, HER2, PTEN, STK11 and TP53) and screened for genetic variations. Sixty-four single nucleotide polymorphisms (SNPs) were identified. Nine of the coding SNPs were non-synonymous, of which four were located in gene regions conserved across four species. Three of the non-synonymous SNPs might be damaging according to PolyPhen predictions. One of the indels identified has previously been associated with CMTs. Because of the founder effects, genetic drift and inbreeding in many dog breeds the allele frequencies of the genes studied are likely to vary significantly between breeds and contribute to the considerable difference in genetic risk associated with cancer.     

Fractal dimension of canine mammary gland epithelial tumors on cytologic smears

BACKGROUND: Fractal geometry is a tool that can be used for describing, modeling, analyzing, and processing irregular and complex figures. Past investigations in medicine have revealed that fractal analysis could also be applied in tumor pathology to characterize irregular boundaries of the nuclei of tumor cells. OBJECTIVE: The aim of this study was to define whether the fractal dimension parameter could be used on cytologic specimens to differentiate benign from malignant canine mammary gland epithelial tumors. METHODS: The fractal dimension of nuclear surface was determined by computer-assisted morphometry on Hemacolor-stained cytologic smears obtained by fine needle aspiration of normal canine mammary gland epithelial cells, and cells in mammary adenomas, tubulopapillary carcinomas, solid carcinomas, and anaplastic carcinomas. Data were analyzed by Mann-Whitney U test. RESULTS: Significant differences (P <.001) were observed in mean fractal dimension among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for the fractal dimension between solid carcinomas and anaplastic carcinomas). CONCLUSION: The morphometric parameter, fractal dimension, could help in the diagnostic discrimination between benign and malignant canine mammary gland epithelial tumors on cytologic specimens.     

Canine mammary carcinomas: influence of histological grade,vascular invasion,proliferation, microvessel density and VEGFR2 expression on lymph node status and survival time

Spontaneous invasive non‐inflammatory canine mammary carcinomas (CMC) and their regional lymph nodes (LN) were analysed (n = 136). Histological grade (HG) and vascular invasion (VI) in the tumours and lymph node status were recorded. Proliferation index (PI), microvessel density (MVD) and vascular endothelial growth factor receptor 2 (VEGFR2) expression were estimated using anti‐proliferating cell nuclear antigen (PCNA), anti‐von Willebrand factor and anti‐Flk‐1, respectively. Eighteen months follow‐up was performed (34 bitches). Tumours of different grades showed differences regarding PI, Flk‐1/integrated optical density (Flk‐1/IOD) and MVD. Every feature showed significant association with LN status through bivariate analyses. From multivariate analyses, VI and Flk‐1/IOD were selected to predict LN status. Data revealed that the probability of a CMC‐bearing bitch to remain alive at 1, 4, 5 and 14–18 months was 0.91, 0.87, 0.81 and 0.77, respectively. Besides LN status, VI was the only feature positively correlated with survival time, although a trend to shorter survival of animal patients bearing high expressing VEGFR2 CMC was noted.     

Immunohistochemical expression of calretinin in canine testicular tumours and normal canine testicular tissue

Calretinin is a calcium-binding protein expressed abundantly in the central and peripheral neural tissues. It has been demonstrated to be a valuable marker in human testicular neoplasia. The immunohistochemical expression of calretinin has been studied in 102 samples of normal (n=25) and three different neoplastic canine testicular tumours (n=77). In normal canine testis, calretinin expression was restricted to Leydig and Sertoli cells of the testis. In tumour tissues, calretinin expression was detected in all tumours investigated (interstitial cell tumours, seminoma, and Sertoli cell tumours), with a cytoplasmic and nuclear pattern of cellular distribution. The present work reports, for the first time, calretinin immunohistochemical expression in normal and neoplastic canine testis.