Cytologic differentiation of benign from malignant canine mammary tumors

Cytologic and histologic examination of 91 canine mammary masses was performed by two cytologists and two histopathologists. Ten important cytologic criteria of malignancy for canine mammary tumors were identified. A cytologic grading system for differentiation of benign from malignant mammary tumors was proposed using these criteria. With this system, approximately one fourth of the malignant mammary tumors were given a concordant cytologic diagnosis. Approximately one-half of the benign masses were given a concordant cytologic diagnosis by the two cytologists. One-half of all the tumors examined were given inconclusive cytologic diagnoses by both cytologists. The cytologic identification of spindle cells did not differentiate complex and mixed mammary tumors from simple tumors. Only five of the animals studied died of mammary cancer, precluding a critical analysis of the cytologic criteria for prediction of cancer mortality.     

Occurrence and expression of p53 suppressor gene and c‐Myc oncogene in dog eyelid tumors

Purpose To detect the occurrence and expression of the suppressor gene p53 and of the oncogene c‐Myc in eyelid tumors of dogs using the PCR, RT‐PCR, PCR‐ELISA and RT‐PCR‐ELISA techniques. These genes have not been described in dog eyelid tumors before. Methods Nine samples of eyelid or third eyelid epithelial tumors were obtained from the archives of the Department of Veterinary Pathology. Tumor diagnosis was confirmed by evaluation of hematoxylin‐eosin stained sections, and immunohistochemistry for cytokeratin AE1/AE3 and vimentin V9. A canine mammary tumor was used for positive control. Agarose gel electrophoresis, PCR‐ELISA and RT‐PCR‐ELISA were used to detect p53 and c‐Myc genes. Results The occurrence of p53 was detected in most of the eyelid tumors and third eyelid tumors studied (88.8%, n = 8) and was expressed in 75% of the positive samples, as indicated by ELISA. The c‐Myc gene was found in 77.7% (n = 7) of the samples and was expressed in eight samples. Conclusions Eyelid and third eyelid tumors of dogs express both the p53 and the c‐Myc genes as shown by PCR and RT‐PCR. However, PCR ELISA and RT‐PCR ELISA were more efficient in assessing occurrence and expression of these genes because they identified amplified products that were not detected by agarose gel electrophoresis.     

Routine hematology and clinical chemistry in female dogs with benign and malignant mammary tumors

Routine hematological and serum biochemical screening was done in 61 female dogs with benign mammary tumors and 51 female dogs with malignant mammary tumors. Most parameters were not significantly different from age-matched female controls; moreover, no significant difference could be observed between animals with benign and malignant tumors. It is concluded that routine hematology and biochemistry offer little diagnostic or prognostic benefit in female dogs with mammary tumors.     

Immunohistochemical detection of Mdm2 and p53 in feline mammary gland tumors

The objective of this study was to evaluate nuclear reactivity of Mdm2 and p53 proteins by immunohistochemical means in feline mammary gland tumors; 12 adenomas which included 6 adenomatous lesions obtained from the tissue adjacent to adenocarcinomas, and 22 adenocarcinomas. Seven adenomas and 18 adenocarcinomas showed moderate or marked Mdm2 reactivity. Sixteen adenocarcinomas showed moderate to marked p53 reactivity, but 9 adenomas showed none. Discordant Mdm2 overexpression was found in 5 adenomas and 3 adenocarcinomas, although co-overexpression of Mdm2 and p53 was found in 15 adenocarcinomas. These results suggest that nuclear overexpression of Mdm2 is present in the tumors of early stage without p53 overexpression and related to feline mammary gland tumorigenesis. Nuclear overexpression of p53 is more frequent in adenocarcinomas, but not in adenomas.     

Expression of maspin in mammary gland tumors of the dog

Maspin is a serine protease inhibitor that inhibits tumor invasion and metastasis in human breast cancer and is consistently expressed by mammary myoepithelial cells (MECs). To analyze the value of maspin as a marker of the MEC layer of the normal and tumoral canine mammary gland, the immunohistochemical expression of maspin was studied in formalin-fixed tissues from 55 benign and malignant tumors (40 tumors also contained the surrounding normal mammary gland) using a commercially available monoclonal antibody. Periacinar and periductal MECs of all 40 normal mammary glands were stained by the anti-human maspin monoclonal antibody, and immunoreactivity was observed in the nucleus and cytoplasm of these cells. In addition, maspin was found in 53 (98%) of the tumors studied, reacting with the MECs in 100% of benign tumors and 93% of malignant tumors and to the epithelial cells of 16% of benign and 73% of malignant tumors. In the MEC compartment, immunoreactivity was observed in the cytoplasm of hypertrophic MECs, fusiform MECs, stellate MECs, rounded (myoepithelial) cells, and chondroblasts. In the epithelial cell compartment, immunoreactivity was observed in the cytoplasm of cells with and without squamous differentiation. Stromal myofibroblasts were unreactive. Maspin appears to be a very sensitive marker of the normal and neoplastic myoepithelium that, contrary to smooth muscle differentiation markers, does not stain stromal myofibroblasts. In addition, a subset of neoplastic epithelial cells reacted with the maspin antibody. The relationship between maspin expression in different cellular compartments of canine mammary carcinomas and the biologic aggressiveness of the disease remains to be elucidated.     

Comparison of B-mode and Doppler ultrasonographic findings with histologic features of benign and malignant mammary tumors in dogs

OBJECTIVE: To compare and correlate B-mode and color Doppler ultrasonographic characteristics with the histologic findings of benign and malignant mammary tumors in dogs. STUDY POPULATION: 49 mammary tumors in 26 dogs. PROCEDURES: Before excision, tumors were evaluated via B-mode and color Doppler ultrasonography to assess size, echogenicity, echopattern, acoustic transmission, invasiveness, and vascularity. Paraffin-embedded microsections of the tumors were stained with H&E and examined for presence of necrosis, cysts, cartilage, bone, mineralization, invasion of surrounding tissue, and tissue heterogeneity. To assess vascularity, the number and distribution of vessels that were stained by the Verhoeff van Gieson technique were recorded. RESULTS: Tumor echogenicity and echopattern on ultrasonographic images correlated with tissue heterogeneity detected histologically. Acoustic enhancement was correlated with the presence of necrotic or cystic areas. Tumor invasion into surrounding tissues as determined ultrasonographically did not correlate with the histologic findings. There was a significant correlation between the number of detected vessels and distribution of flow within the tumors determined via ultrasonographic and histologic examinations. CONCLUSIONS AND CLINICAL RELEVANCE: In canine mammary tumors, ultrasonographic characteristics appear to be correlated with histopathologic changes. Data suggest that ultrasonography may have an important role in the evaluation of mammary tumors in dogs, particularly in the evaluation of tissue composition and tumor vascularity.     

TP53 mutations in canine brain tumors

The p53 tumor suppressor gene (TP53) is the most frequently altered gene in human cancer. Mutation of the gene has been shown to be an important mechanism of p53 pathway inactivation in a variety of human brain tumors, particularly those of astrocytic origin. Genomic DNA from a series of 37 glial and 51 nonglial canine brain tumors was sequenced to determine the frequency of TP53 gene mutations involving exons 3-9. Exonic mutations were found in 3 of 88 tumors (3.4%) and specifically in 1 of 18 astrocytic tumors (5.5%). This is markedly lower than that reported in comparable human tumors, suggesting that alternative mechanisms of p53 inactivation are likely to be present if p53 function contributes significantly to oncogenesis in canine brain tumors.     

Metallothionein expression in benign and malignant canine mammary gland tumours

The presence of metallothioneins (MTs) were demonstrated immunohistochemically using a monoclonal antibody (E9) against a conserved epitope of I and II isoforms in canine mammary tumours. In a semiquantitative analysis MT expression in the tumour cells was observed in 54/54 cases of benign and 32/40 malignant mammary neoplasms. A statistically significant difference at the level of P<0.01 was observed for MT expression between benign and malign mammary tumours in terms of immunoreactivity score. It is concluded that immunohistochemically demonstrated MT expression is significantly associated with benign canine mammary tumours.     

Immunohistochemical evaluation of p53 expression with different antibodies in malignant canine tumours with or without p53 gene mutation

Six monoclonal antibodies and a polyclonal antibody (CM1) were used to investigate the overexpression of p53 protein by immunohistochemistry (IHC) in six sarcomas and 21 mammary carcinomas from 27 dogs. IHC was compared with p53 gene mutation analysis performed on the same samples. Only the monoclonal PAb240, PAb421 and the CM1 antibodies were able to detect expression of canine p53 protein. CM1 was found to give the highest concordance (8/11) between positive expression of the p53 protein by IHC and the presence of a gene mutation. In the samples that were negative for p53 expression by IHC, but contained a p53 gene mutation according to DNA analysis, the mutation often affected the epitopes that could have been recognized by these antibodies. Only one out of 16 tumours without a p53 gene mutation had a weakly positive IHC result. These findings indicate that in these two types of canine tumours, IHC – particularly with CM1 – can detect many alterations in p53 expression owing to a gene mutation. False‐positive results were very infrequent.     

Tumour suppressor gene p53 mutation in a case of haemangiosarcoma of a dog

Haemangiosarcomas of dogs were analysed by molecular genetic techniques. Regions of the tumour suppressor gene p53, including the well-known tumour hot spots (codons 175, 245, 248, 249, 273 and 282) were screened. A 24 bp deletion was detected in exon 5 of the gene.     

Cytomorphologic differentiation of benign and malignant mammary tumors in fine needle aspirate specimens from irradiated female Sprague-Dawley rats.

BACKGROUND: Fine needle aspiration (FNA) offers a rapid and minimally invasive means to distinguish malignant from benign neoplasms. However, few studies have been published regarding the cytopathology of mammary tumors in rats despite widespread use of the rat model for breast cancer formation and inhibition. OBJECTIVE: The purpose of this study was to determine the diagnostic accuracy of FNA cytology and to develop distinguishing cytologic criteria for the diagnosis of radiation-induced benign and malignant mammary tumors in rats. METHODS: In a study of radiation-induced mammary carcinogenesis, 100 Sprague-Dawley rats with cutaneous masses were randomly chosen for FNA. The aspirates were smeared, fixed, and stained with a modified Papanicolaou procedure for diagnostic evaluation. Cytologic and histologic diagnoses (benign vs malignant) were compared, and diagnostic accuracy was calculated using the histologic diagnosis as the criterion standard. FNA smears were scored semiquantitatively on a scale of 1-4 for cellularity, atypia, nuclear size, chromatin pattern, nuclear membrane thickness, nucleoli, and mitoses. The background was evaluated for necrosis, hemorrhage, inflammation, and mucosecretory material. Cytomorphologic features were compared statistically between benign and malignant tumors, based on the histologic diagnosis. RESULTS: The sensitivity of FNA was 92.3% and specificity was 89.4% for the detection of malignancy. However, 14% of specimens, all fibroadenomas by histology, had insufficient cells for cytologic evaluation, for an overall accuracy rate of 78.0%. Malignant tumors had significantly higher scores for all cytomorphologic features, and were significantly more likely to contain cell clusters and necrotic debris. CONCLUSIONS: FNA is an accurate method for differentiating benign and malignant rat mammary tumors.     

Presence of p53 mutations in feline neoplasms

A region from exon 4 to 8 of the tumour suppressor gene p53 was analysed in 60 feline tumours (30 fibrosarcomas, seven malignant histiocytomas, three lymphosarcomas, five basal cell tumours, five squamous cell carcinomas, two adenocarcinomas of tubular skin glands, one undifferentiated carcinoma of the skin, seven mammary carcinomas). Missense mutations were detected in two fibrosarcomas, one malignant fibrous histiocytoma, the undifferentiated carcinoma of the skin and one mammary carcinoma. One nonsense mutation was detected in one fibrosarcoma and one deletion/frameshift-mutation was observed in one squamous cell carcinoma.     

Immunohistochemical analysis of c-yes and c-erbB-2 oncogene products and p53 tumor suppressor protein in canine mammary tumors

In order to evaluate the involvement of c-yes and c-erbB-2 oncogene products, and p53 tumor suppressor protein in canine mammary neoplastic lesions, sections of archived paraffin-embedded samples of 79 mammary tumors were analyzed immunohistochemically using antibodies against human c-yes p62 and c-erbB-2 products and p53. These 79 tumors were divided into 2 groups: 32 benign (2 adenosis, 7 simple adenomas, 14 complex adenomas, and 9 benign mixed mammary tumors) and 47 malignant tumors (26 simple adenocarcinomas, 7 complex adenocarcinomas, 5 solid carcinomas, 2 sclerosing carcinomas, 6 malignant mixed mammary tumors, and 1 malignant myoepithelioma). As a result of immunostaining, 40.6% (13/32) of the benign tumors and 21.3% (10/47) of the malignant tumors expressed the c-Yes oncogene product, ErbB-2 expression was detected in 50% (16/32) of the benign tumors and in 19.1% (9/47) of the malignant tumors. P53 expression was detected in 16% (4/25) of the benign tumors and in 30.6% (11/36) of the malignant tumors. Co-expression of c-Yes and ErbB-2, ErbB-2 and p53, and all 3 products was detected in 6, 1 and 7 tumors, respectively.     

Evaluation of satraplatin in dogs with spontaneously occurring malignant tumors

Background: Satraplatin is the 1st orally bioavailable platinum anticancer drug. Objective: Our objectives were to evaluate efficacy in vitro against a canine cancer cell line, to determine the maximally tolerated dose (MTD) of satraplatin in tumor‐bearing dogs, to identify the dose‐limiting and other toxicities in dogs, and to record pharmacokinetics (PK). Animals: Dogs with macro‐ or microscopic malignant neoplasia. Methods: D17 canine osteosarcoma cells first were evaluated in a clonogenic survival assay. Then, dogs with a diagnosis of malignant neoplasia were prospectively entered in standard 3 + 3 cohorts. Additional patients were entered at the MTD to assess efficacy. Total and free platinum (by ultrafiltrate) concentrations were determined with inductively coupled plasma mass spectroscopy. Results: Satraplatin inhibited clonogenic survival in vitro at clinically relevant and achievable concentrations. Twenty‐three dogs were treated, 14 with PK evaluation. The MTD was 35 mg/m²/d for 5 days, repeated every 3–4 weeks. Bioavailability was 41%. PK variables (mean ± SD) at the MTD included T_max 1.8 (± 0.7) hours, C_max 72 (± 26) ng/mL, area under concentration (AUC)_{0–24 h} 316 (± 63) h × ng/mL, and MRT 7 (± 1.3) hours. Higher AUC after the 5th versus the 1st dose suggested drug accumulation. Interestingly, platelets consistently reached nadir sooner than did neutrophils (day 14 versus 19). Myelosuppression was dose‐limiting and gastrointestinal toxicity was mild. Conclusions and Clinical Importance: Satraplatin was well tolerated in tumor‐bearing dogs, thus warranting further investigation in a phase II trial.     

Cytomorphologic differentiation of benign and malignant mammary tumors in fine needle aspirate specimens from irradiated female Sprague–Dawley rats

Background: Fine needle aspiration (FNA) offers a rapid and minimally invasive means to distinguish malignant from benign neoplasms. However, few studies have been published regarding the cytopathology of mammary tumors in rats despite widespread use of the rat model for breast cancer formation and inhibition.
Objective: The purpose of this study was to determine the diagnostic accuracy of FNA cytology and to develop distinguishing cytologic criteria for the diagnosis of radiation-induced benign and malignant mammary tumors in rats.
Methods: In a study of radiation-induced mammary carcinogenesis, 100 Sprague–Dawley rats with cutaneous masses were randomly chosen for FNA. The aspirates were smeared, fixed, and stained with a modified Papanicolaou procedure for diagnostic evaluation. Cytologic and histologic diagnoses (benign vs malignant) were compared, and diagnostic accuracy was calculated using the histologic diagnosis as the criterion standard. FNA smears were scored semiquantitatively on a scale of 1–4 for cellularity, atypia, nuclear size, chromatin pattern, nuclear membrane thickness, nucleoli, and mitoses. The background was evaluated for necrosis, hemorrhage, inflammation, and mucosecretory material. Cytomorphologic features were compared statistically between benign and malignant tumors, based on the histologic diagnosis.
Results: The sensitivity of FNA was 92.3% and specificity was 89.4% for the detection of malignancy. However, 14% of specimens, all fibroadenomas by histology, had insufficient cells for cytologic evaluation, for an overall accuracy rate of 78.0%. Malignant tumors had significantly higher scores for all cytomorphologic features, and were significantly more likely to contain cell clusters and necrotic debris.
Conclusions: FNA is an accurate method for differentiating benign and malignant rat mammary tumors.