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.     

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.     

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.     

Immunophenotyping lymphomas in dogs: a comparison of results from fine needle aspirate and needle biopsy samples

The reliability of utilizing cytologic samples for immunophenotyping canine lymphomas was evaluated by a systematic comparison of results from fine needle aspirate (FNA) to needle biopsy specimens stained by immunochemical methods. The specific reactivity of a selected panel of 12 antibodies to cell surface markers and intermediate filaments was assessed in 11 dogs by comparing cytologic to histologic samples. There was excellent correlation of results between immunostained cytologic and histologic samples. FNA sampling is a simple, noninvasive method for determining the lymphoid phenotype in canine lymphoma. In addition, this technique may be useful as a cytodiagnostic aid in differentiating lymphoid from non-lymphoid tumors and in assessing neoplastic vs. reactive or hyperplastic processes.     

Identification of microfilaria in a mammary mass aspirate from a female dog

A filarial nematode larva (microfilaria) was found in an aspirate of a mammary mass from a dog imported to Saskatchewan from Myanmar. This was an unusual location for a microfilaria and numerous filarial nematodes infecting dogs in Asia had to be considered. This report describes the laboratory diagnosis of canine heartworm disease in this dog.     

Conventional and Doppler ultrasound for the differentiation of benign and malignant canine mammary tumours

Objectives : The aim of this study was to evaluate the sensitivity of conventional and Doppler ultrasound for differentiation of benign and malignant mammary tumours in female dogs. Methods : Mammary tumours were evaluated from 60 animals and divided into two distinct groups, group 1 (benign tumours) and group 2 (malignant tumours). The tumours were assessed by conventional ultrasound, Doppler ultrasound mode, histopathology and immunohistochemical detection of vascular endothelial growth factor. Results : Conventional ultrasound examination was found to be ineffective in separating tumours into the two experimental groups. Similarly, using colour‐flow Doppler ultrasound, no correlation was found between the presence of vascularisation and its characteristics between the two groups. Triplex Doppler ultrasound yielded average maximum velocities of 28·71 cm/s for malignant and 19·91 cm/s for benign tumours, which were significantly different (P=0·01). For vascular endothelial growth factor, an average score of 2·22 was found for group 2 and 1·66 for group 1 (P=0·03). Positive correlations were found between vascular endothelial growth factor and presence of vascularisation (P=0·04 and r=0·3658) and between vascular endothelial growth factor and maximum velocity (P=0·03 and r=0·3913). Clinical Significance : Doppler evaluation may be used to predict malignancy of mammary tumours in bitches.     

p53 gene mutations occurring in spontaneous benign and malignant mammary tumors of the dog

Sixty-three cases of benign and malignant canine mammary tumors were analyzed to define the alteration of exons 5-8 for the p53 tumor suppressor gene using polymerase chain reaction direct sequence analysis with paraffin-embedded tissues. Four missense mutations were found in 38 benign mammary tumors (11%), and five missense (one tumor had two missense mutations) and one nonsense mutations were found in 25 mammary carcinomas (20%). These data suggest that the p53 gene alterations might be initiated at an early stage of canine mammary carcinogenesis and p53 mutations might be associated with malignancy. However, there was no evidence of any relationship between the p53 alterations and the histologic types of tumors or breeds of dogs.     

Application of rapid CD3 immunophenotype analysis and argyrophilic nucleolar organizer region (AgNOR) frequency to fine needle aspirate specimens from dogs with lymphoma

Determinations of CD3 immunoreactivity (CD3-IR) and argyrophilic nucleolar organizer region (AGNOR) frequency from fine needle aspirate (FNA) samples were compared with those from formalin-fixed, paraffin-embedded surgical biopsy samples in 51 dogs with lymphoma. Both CD3-IR (using a rapid EPOS polymer system) and AGNOR techniques were readily applied to FNA samples. CD3-IR from FNA samples matched those of histologic samples with 100% concordance. A linear relationship (r=0.981; P<0.001) was found between AGNOR frequency obtained from FNA samples and those obtained from surgical biopsy samples. Application of the techniques presented here should allow clinically relevant information to be procured rapidly and inexpensively. As CD3-IR and AGNOR frequency have been shown to be predictive of response to combination chemotherapy in dogs with lymphoma, such information could be used to better educate clients as to the likelihood of achieving meaningful responses, as well as allowing prospective tailoring of individual treatments in future trials prior to initiating therapy.     

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.     

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.     

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.     

Immunohistochemistry for parathyroid hormone-related protein (PTHrP) in benign and malignant mammary mixed tumors of dogs with and without hypercalcemia

We evaluated the expression of parathyroid hormone-related protein (PTHrP) by immunohistochemistry in eight benign and malignant mammary mixed tumors of dogs with (n = 4) and without (n = 4) hypercalcemia. Positive immunoreactive staining for PTHrP was observed in all four tumors from hypercalcemic dogs. The mammary tumors from 2 of the 4 normocalcemic dogs stained positively for PTHrP, but the numbers of immunoreactive cells and intensity of the immunoreaction were less than in the hypercalcemic dogs. In the other 2 tumors without hypercalcemia, the tissue samples were negative for PTHrP.     

Granular cell lesions in the distal female reproductive tract of aged Sprague-Dawley rats

During the review of a rat carcinogenicity study, a spectrum of granular cell lesions was recognized in the distal female reproductive tract. To verify the diagnoses, cell populations of nine granular cell alterations of the cervix or vagina were characterized immunohistochemically and four were evaluated ultrastructurally. Immunoreactivity was demonstrated in 8/9 cases with S100 protein, 6/9 cases with neuron-specific enolase, and 7/9 cases with Leu-7. Granular cells were negative for smooth muscle-specific actin and calretinin. The immunohistochemical profile of these lesions was similar to that previously reported in other species, including humans. Ultrastructurally, as expected many lysosomal bodies were present in the cytoplasm of granular cells in all specimens evaluated. Based on the detailed evaluation of a series of lesions, we adopted the following diagnostic criteria and nomenclature for the granular cell changes of the female reproductive tract of rats. Granular cell aggregates were non-space-occupying lesions composed of clusters of typical granular cells. Benign granular cell tumors were space occupying lesions that typically contained prominent interstitial collagen and were either discrete masses or were difficult to discern from the surrounding tissues. Some benign tumors also contained foci of spindle cells with decreased granularity. Malignant tumors exhibited pleomorphism and an increased nucleus: cytoplasm ratio morphologically but had the same biologic behavior as the benign tumors. We applied these diagnostic criteria during the review of controls from 9 carcinogenicity studies. Up to 23% of control females in those carcinogenicity studies had granular cell lesions that could be classified into one of the three categories. Granular cell lesions appear to be common in the cervix/vagina of the Sprague-Dawley rat, and tumors may develop from granular cell aggregates.     

Expression patterns of the slit subfamily mRNA in canine malignant mammary tumors

Slit, a secreted protein, functions as a chemorepellent factor in axon guidance and neuronal migration and as an inhibitor in leukocyte chemotaxis. In humans, slit2 protein attracts endothelial cells and promotes tube formation in the tumor angiogenic mechanism. In this study, we cloned a part of the canine slit subfamily and examined the expression of slit subfamily mRNAs in 3 normal canine mammary glands and 11 mammary tumor samples by RT-PCR. The cloned part of the slit gene sequences showed high similarity to those of the human, mouse, and rat. The mRNAs were expressed at low levels in the normal mammary gland. The expression levels of slit1 mRNA were low in both the normal and tumor tissues. In contrast, the expression of slit2 mRNA increased in most of the malignant mammary tumors, and an increase in slit3 mRNA expression was observed in 2 of the malignant mixed tumors. These results suggest that the expression of slit2 plays an important role in tumor angiogenesis in canine mammary gland tumors and that slit2 can be a putative marker for malignancy diagnosis of these tumors.     

Canine mammary gland tumours; a histological continuum from benign to malignant; clinical and histopathological evidence*

This study describes the clinical and histopathological findings in dogs with mammary gland tumours, and compares the histopathological and clinical evidence consistent with progression from benign to malignant to human breast cancer epidemiology. Clinical and histopathological data on 90 female dogs with 236 tumours was included. Dogs with malignant tumours were significantly older than dogs with benign tumours (9.5 versus 8.5 years), P = 0.009. Malignant tumours were significantly larger than benign tumours (4.7 versus 2.1 cm), P = 0.0002. Sixty‐six percent had more than one tumour, and evidence of histological progression was noted with increasing tumour size. Dogs with malignant tumours were significantly more likely to develop new primary tumours than dogs with benign tumours, P = 0.015. These findings suggest that canine mammary tumours progress from benign to malignant; malignant tumours may be the end stage of a histological continuum with clinical and histopathological similarities to human breast carcinogenesis.     

Ultrasound-guided fine needle aspiration in the diagnosis of peripheral nerve sheath tumors in 4 dogs

Ultrasound-guided fine needle aspiration was used in establishing the diagnosis in 4 cases of malignant peripheral nerve sheath tumor. Sonographic and cytologic characteristics are discussed. Because of its availability and ease of use, axillary ultrasonography with fine needle aspiration can be an initial diagnostic step for suspected brachial plexus tumors.