Evaluation of microsatellite instability in urine for the diagnosis of transitional cell carcinoma of the lower urinary tract in dogs

Background: The accumulation of frame‐shift mutations in microsatellites (MS), termed microsatellite instability (MSI), is associated with certain tumors. MSI and its detection in urine samples has been used to aid in the detection of human bladder cancer. Hypothesis: Evaluation of MSI in urine is a useful assay test for diagnosis of transitional cell carcinoma (TCC) in dogs and is more specific than the commercially available, veterinary bladder tumor analyte (V‐BTA) test. Animals: Seventy‐three dogs: healthy controls (n= 21), proteinuric (n= 12), lower urinary tract disease excluding TCC (n= 17), and TCC (n= 23). Methods: Prospective observational study. Urine samples collected from each animal were evaluated for MSI and using the V‐BTA. For MSI detection, 22 MS sequences were polymerase chain reaction amplified from urine and blood, subjected to capillary electrophoresis, and the MS genotypes were compared. Aberration in ≥15% of MS was considered indicative of MSI. Results: MSI was detected in 11 of 23 (48%) urine samples from dogs with TCC. MSI was also detected in 12 of 50 (24%) of the control animals, including 29, 16, and 24% of healthy, proteinuric, and lower urinary disease dogs, respectively. In this population, sensitivity and specificity of MSI analysis was 48 and 76%, respectively, compared with 83 and 64%, respectively, for the V‐BTA test. Conclusions: MS analysis as performed in this study is not useful in the diagnosis of TCC.     

Immunohistochemical assay for detecting estrogen receptors in canine mammary tumors.

OBJECTIVE: To determine the estrogen receptor (ER) content of canine mammary gland tumors by use of immunohistochemical (IHC) examination of formalin-fixed sections. SAMPLE POPULATION: 21 mammary gland tumors from 20 adult dogs. PROCEDURE: ER were detected in formalin-fixed tissues, using an avidin-biotin alkaline phosphatase IHC assay and were quantified on fresh-frozen tumor samples, using a modified dextran-coated charcoal (DCC) assay. RESULTS: 7 of 21 tumors had visually detectable nuclear ER by use of IHC staining, whereas 8 of 21 tumors were positive for ER by use of the DCC assay. The ER-positive cells in 5 IHC-positive tumors were epithelial cells with histologic criteria of early malignancy. The remaining 2 ER-positive tumors detected by use of IHC had ER-positive mast cells within areas of connective tissue around the tumor. CONCLUSIONS: Immunohistochemistry is an additional method for detection of ER in canine mammary tumors. The major advantage of this type of assay is that it may be performed on formalin-fixed tissues, and individual ER-positive cells may be identified. Discovery of ER-positive mast cells by use of IHC is of concern, particularly if the ER status of a tumor is based on DCC results alone. CLINICAL RELEVANCE: Because most canine mammary tumors are fixed in formalin prior to histologic evaluation, an IHC assay that identifies ER-positive cells is desirable. Adjunctive antiestrogen therapy could be administered to dogs with ER-positive tumors.     

Novel variations and loss of heterozygosity of BRCA2 identified in a dog with mammary tumors

OBJECTIVE: To establish novel polymorphic markers for analysis of loss of heterozygosity (LOH), so as to study the possible involvement of BRCA2 in mammary tumors obtained from dogs. SAMPLE POPULATION: Blood samples, mammary gland specimens, or mammary tumors from 3 tumor-bearing dogs and 10 tumor-free dogs. PROCEDURES: Nucleotide sequence analysis was performed with a DNA autosequencer. Loss of heterozygosity analysis was performed for markers established in the present study. The expression level of canine BRCA2 was quantified by real-time PCR analysis. RESULTS: 3 novel microsatellite markers with high heterozygosity rates (> 50%) were established, and the previously reported marker for canine BRCA2 gene locus was improved. These markers were used for the analysis of DNA from formalin-fixed and paraffin-embedded samples. By use of these markers, LOH in canine BRCA2 was identified as a result of recombination. In mammary tumor DNA that corresponded to the LOH-positive dog, the level of canine BRCA2 expression was decreased compared with that of nonneoplastic mammary gland tissue; the open reading frame contained 4 missense variations, 1 insertion variation, and 1 silent variation, some of which were localized to functional domains. CONCLUSIONS AND CLINICAL RELEVANCE: 3 novel polymorphic markers were developed for LOH analysis of canine BRCA2 and identified a dog with LOH with some variations in the functional domains. These markers could be useful for assessing the relevance of BRCA2 variation in mammary tumors of dogs.     

Results of hyperamplification of centrosomes in naturally developing tumors of dogs.

OBJECTIVE: To evaluate results of centrosome hyperamplification in naturally developing tumors of dogs. SAMPLE POPULATION: Tumor specimens from 9 dogs with tumors (rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myxosarcoma, and mammary gland tumor) and 2 canine osteosarcoma cell lines. PROCEDURE: 3 antibodies for centrosome proteins (ie, anti-gamma-tubulin, anti-BRCA1, and anti-pericentrin) were used for immunohistochemical analysis. Double immunostaining for centrosomes was used to confirm the specificity of these antibodies for centrosomes. Mutational analysis of the canine p53 gene was carried out by polymerase chain reaction-single-strand conformation polymorphism analysis, and expression of canine MDM2 protein was evaluated by use of immunohistochemical analysis, using anti-MDM2 antibody. RESULTS: Immunohistochemical analysis of dog osteosarcoma cell lines with apparent aneuploidy revealed frequent hyperamplification of centrosomes in the osteosarcoma cell lines. Similar hyperamplified centrosomes were detected in the tumor tissues from all of the 9 tumors. The frequency of cells with hyperamplified centrosomes (3 to 20/cell) in each tumor tissue ranged from 9.50 to 48.1%, whereas centrosome hyperamplification was not observed in normal lymph nodes from these dogs. In 8 of the 9 tumors, mutation of p53 gene or overexpression of MDM2, or both, was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Various types of naturally developing tumors in dogs often have hyperamplification of centrosomes associated with chromosome instability. Hyperamplification of centrosomes is a novel tumor marker for use in cytologic and histologic examinations of clinical specimens obtained from dogs.     

Correlation between the histopathological diagnosis by AgNOR count and AgNOR area in canine mammary tumors

Background: Mammary tumors are the most common type of tumor in female dogs. The histopathological diagnosis is usually made by a hematoxylin-eosin (HE) staining of the tumor, which then requires a pathologist's judgment for assessment of malignancy. The purpose of this study was to investigate an alternative silver staining of some argyrophilic nucleolar organizer regions (AgNOR) for improving the diagnostic accuracy with mammary tumors.
Hypothesis: There is a correlation between the histopathological diagnosis by AgNOR count and AgNOR area in canine mammary tumors.
Animals: Seventy-three canine mammary tumors from 33 female dogs.
Materials and Methods: The AgNOR staining was evaluated retrospectively in 73 canine mammary tumors with a parallel HE staining as a "Gold Standard." Both a quantitative manual counting method and a qualitative computerized morphometric method were tested.
Result: The result from both methods indicated a clinically relevant difference in the mean values of the AgNOR in the following 4 categories: malignant, benign, hyperplastic, and normal mammary tissue. The counting method was superior, with 89% of the cases given a correct diagnosis of a malignant or a nonmalignant canine mammary tumor. The 2 methods were then compared to test their ability to classify the tumors correctly. Again, the counting method was the most reliable method, with a sensitivity of 80% and a specificity of 76% when the upper 50% of the AgNOR counts were presumed malignant.
Conclusion and Clinical Importance: The results indicated that an AgNOR test could be an aid to pathologists as a prognostic indicator or to assist them in deciding between a benign or a malignant diagnosis in questionable cases.     

The activity of matrix metalloproteinases (MMPS) and tissue inhibitors of metalloproteinases (TIMPs) in mammary tumors of dogs and rats

We conducted zymography for detecting the activity of matrix metalloproteinases (MMPs) and reverse zymography for the activity of tissue inhibitors of metalloproteinases (TIMPs) in canine spontaneous and rat 7, 12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor tissues. The activities of MMPs of canine mammary tumors were quite higher than those of the rat chemically induced tumors. The activities of MMPs were significantly higher in malignant tissues than in benign ones of canine tumors, whereas the activity of only MMP-2 was higher in both benign and malignant rat tumors compared to normal tissues. There were no differences of MMPs activities between benign and malignant rat tumors. The results of reverse zymography indicated that the activities of TIMP-1, -2 and -3 were strikingly higher in rat tumors than in canine tumors. The activities were higher in malignant tissues than in benign ones of dogs, and higher in tumor tissues than in normal mammary tissues of rats. The results of film in situ zymography for tissue localization of gelatinolytic activity showed that the digested area was more extended in malignant tumors than in benign ones of dogs. However, the area was similarly extended in both benign and malignant rat tumors. These results may indicate that the canine spontaneous malignant mammary tumors possess more aggressive nature than the rat chemically induced counterpart, resulting from the high level of MMPs and low level of TIMPs activities of the tumor tissues.     

The Prognostic Significance of Angiogenesis in Canine Mammary Tumors

The purpose of the study was to determine if neovascularization, a measure of angiogenesis, is correlated with metastasis of mammary tumors in dogs. Forty-six paraffin-embedded tissue blocks of benign and malignant canine mammary tumors obtained from 42 clinical cases at the Iowa State University Veterinary Teaching Hospital were retrieved from the archives of the Department of Veterinary Pathology. Of the dogs with malignant tumors, cases with and without lymph node metastasis were chosen. Neovascularization was quantified by light microscopy on formalin-fixed, paraffin-embedded sections of canine mammary tumors using an avidin biotin immunoperoxidase assay for factor VIII-related antigen. Mean microvessel counts for each group were statistically evaluated using analysis of variance. The mean number of microvessels was highest in the malignant tumors of dogs with lymph node metastasis (44). This number was significantly different from the mean number of microvessels in the benign tumors (28; P = .03) and a trend occurred toward higher microvessel counts in malignant tumors with lymph node metastasis versus malignant tumors of dogs without metastasis (32; P = .1). No significant difference was found between the number of microvessels found in malignant tumors without metastasis versus benign tumors. The trend toward higher microvessel counts in mammary tumors that have metastasized supports the premise that angiogenesis may be an independent and significant prognostic indicator in dogs with malignant mammary tumors, as it is in women with breast cancer.     

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.     

Telomere length and telomerase activity in canine mammary gland tumors

OBJECTIVE: To measure telomere length and telomerase activity in naturally occurring canine mammary gland tumors. SAMPLE POPULATION: 27 mammary gland tumor specimens obtained during resection or necropsy and 12 mammary gland tissue specimens obtained from healthy (control) dogs. PROCEDURE: Telomere length in tissue specimens was measured by use of restriction endonuclease digestion and Southern blot analysis. Telomerase activity was measured by use of a telomeric repeat amplification protocol assay. RESULTS: Telomere length in mammary gland tumors ranged from 11.0 to 21.6 kilobase pairs (kbp; mean +/- SEM, 14.5+/-0.5 kbp) but did not differ among tumor types. Telomeres in mammary gland tumors were slightly shorter than in normal tissue specimens, but telomere length could not be directly compared between groups, because mean age of dogs was significantly different between groups. Age was negatively correlated with telomere length in control dogs but was not significantly correlated with length in affected dogs. Telomerase activity was detected in 26 of 27 mammary gland tumors and in 4 of 12 normal tissue specimens. However, telomerase activity and telomere length were not correlated in tumor specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Telomere length is maintained in canine mammary gland tumors regardless of the age of the affected dog. Measurement of telomere length may be a useful tool for monitoring the in vivo effects of telomerase inhibitors in dogs with tumors.     

PTEN 基因在犬乳腺肿瘤组织中的表达及临床意义

有关酪氨酸磷酸酶基因（Phosphatase and tensin homolog deleted on chromosometen,PTEN）在乳腺肿瘤中的检测在人医早有报道。为了研究PTEN基因在犬乳腺肿瘤组织中的表达情况,笔者运用实时荧光PCR定量检测了38例不同的犬乳腺肿瘤组织（包括15例良性乳腺肿瘤和23例恶性乳腺肿瘤）、4例正常犬乳腺组织。结果发现：PTEN基因在犬恶性乳腺肿瘤组织中表达量明显低于其在良性乳腺肿瘤和正常乳腺组织中的表达量,两者差异极显著（P〈0.001）;PTEN在良性乳腺肿瘤组织中的表达与正常犬乳腺组织相比,差异不显著（P〉0.05）;发生了淋巴结转移的乳腺癌PTEN基因的表达量与未发生转移的乳腺癌组织的表达量间差异亦不显著（P〉0.05）,且PTEN的表达量与肿瘤组织的大小和发病动物年龄无关。结论：PTEN蛋白表达异常可能与乳腺肿瘤发生、发展相关,可考虑作为判断犬乳腺肿瘤生物学行为和预测的指标。     

CHARACTERIZATION OF CANINE SUPERFICIAL TUMORS USING GRAY-SCALE B MODE, COLOR FLOW MAPPING, AND SPECTRAL DOPPLER ULTRASONOGRAPHY—A MULTIVARIATE STUDY

Superficial tumors are not routinely evaluated by two- or three-dimensional diagnostic imaging methods as part of the staging of canine cancer patients, although superficial tumors are readily imaged by ultrasound. The objectives of this study were to characterize the ultrasonographic patterns of superficial tumors and to evaluate whether ultrasound can help discriminate between benign and malignant tumors in dogs. Superficial tumors (n=132) in 86 dogs were evaluated by B mode, color flow mapping, and spectral Doppler ultrasonography. Size, echogenicity, tumor border definition, invasiveness, acoustic transmission, presence and distribution of vascular flow to and within the tumor, as well as perfusion indices were measured. The tumors were classified as lipomas, benign tumors, atypical mammary tumors, and malignant tumors. Multivariate statistics using discriminant analysis was used to determine which parameters may be used to predict the status of the tumor. Tumor echogenicity, border shape, acoustic shadowing, total number of vessels to the tumor and the total flow amount are the parameters that in combination resulted in the lowest classification error (24%), meaning that on average three out of four tumors were correctly classified using these parameters. All the lipomas and atypical mammary tumors were classified correctly by ultrasonography. The results of this study show that ultrasonography has an important role in the evaluation of canine superficial tumors, particularly in the evaluation of tissue homogeneity and tumor vascularity.     

Alpha basic crystallin expression in canine mammary tumors

The aim of this study was to evaluate prognostic and/or diagnostic factors of canine mammary tumors by immunohistochemically analyzing the expression of alpha basic crystallin (αB-c). For this, formalin-fixed, paraffin-embedded blocks of 51 naturally-occurring canine mammary tumors (11 benign and 40 malignant) were used. Tissue from eight normal canine mammary glands were served as a control. Immunohistochemically, in the control mammary tissues, a few luminal epithelial cells were αB-c positive but myoepithelial cells were negative. In benign or simple type malignant tumors, αB-c expression was observed in luminal epithelial cells while the myoepithelial basal cells were negative. In benign or complex type malign tumors, positive staining was predominantly found in the cytoplasm of epithelial cells. Immunoreactivity of αB-c was also observed in neoplastic myoepithelial cells. Statistically, the number of cells immunolabeled with αB-c was found to be significantly different among tissues from normal canine mammary glands, benign lesions, and malignant tumors (p < 0.05). αB-c immunoreactivity was higher in malignant tumors than the control mammary tissues (p < 0.001). Data obtained in the current study revealed a strong association between high expression levels of αB-c and primary mammary gland tumors in canines.     

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.     

Immunohistochemical analysis of cyclin A, cyclin D1 and P53 in mammary tumors, squamous cell carcinomas and basal cell tumors of dogs and cats

The involvement of cyclin A, cyclin D1 and p53 proteins in canine and feline tumorigenesis was analyzed immunohistochemically. In the present study, a total of 176 cases were examined, among which there were 108 canine cases (75 mammary lesions, 16 squamous cell carcinomas and 17 basal cell tumors) and 68 feline cases (43 mammary lesions, 20 squamous cell carcinomas and 5 basal cell tumors). Speckled nuclear staining for cyclin A was observed in 19/38 (50%) canine malignant mammary tumors and 18/37 (48.6%) feline mammary carcinomas, while this was not seen in benign mammary tumors of either dogs or cats. Marked intense nuclear cyclin A staining was seen in 7/16 (43.8%) canine squamous cell carcinomas and 18/20 (90.0%) feline squamous cell carcinomas. Only 3/17 (17.6%) canine basal cell tumors showed slight and scattered staining for cyclin A. Expression of cyclin D1 was very rare in both canine and feline tumors. Nuclear staining of p53 was found in 7/37 (18.9%) feline mammary carcinomas. Intense immunoreactivity for p53 was found in 6/16 (37.5%) canine squamous cell carcinomas and 8/20 (40%) feline squamous cell carcinomas. These results suggest that cyclin A may have a role in the proliferation of canine malignant mammary tumors, feline mammary carcinomas and squamous cell carcinomas of dogs and cats, and p53 may associate with the tumorigenesis of feline mammary carcinomas and squamous cell carcinomas of dogs and cats.     

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.     

Correlation of DNA ploidy to tumor histologic grade, clinical variables, and survival in dogs with mast cell tumors.

By using flow cytometry, a retrospective analysis of the DNA content of 40 primary canine mast cell tumors and seven lymph nodes that contained metastatic mast cell tumor from 44 dogs of various breed, sex, and age was performed on formalin-fixed, paraffin-embedded samples of the tumors and nodes. These samples were chosen according to the following criteria: samples contained sufficient well-preserved tumor tissue in the paraffin block for processing, sufficient patient history data were available, clean and homogeneous cell suspensions were obtained after processing, and interpretable DNA histograms were produced on analysis. The ploidy data obtained were compared with the histopathologic grade, the anatomical site of occurrence, the clinical stage of the tumors, and the survival of the dogs. Over 70% (29/40) of the mast cell tumors were diploid. Three metastatic mast cell tumors in lymph nodes had the same ploidy status as their corresponding primary tumors. In five dogs, mast cell tumors from multiple sites in each dog displayed similar ploidy status. Of 26 dogs evaluated for survival times, 69% (18/26) had diploid tumors and 31% (8/26) had aneuploid tumors. When numbers of diploid versus aneuploid tumors were compared, no significant difference was found between any two grades, clinical stages, or anatomic sites. A significant difference (P = 0.02) was found, however, between aneuploid and diploid tumors when comparing Stage I and non-Stage I disease. The Kaplan-Meier survival plot indicated a tendency towards an increased survival within the first year in dogs with diploid versus aneuploid tumors (P = 0.06).     

Immunohistochemical expression of estrogen receptor beta in normal and tumoral canine mammary glands

To date, two isoforms of estrogen receptors (ER) have been identified, cloned, and characterized from several species, estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta). Although the presence of ERalpha has been demonstrated in normal and tumoral canine mammary tissues, the issue of ERbeta expression has not been addressed in the dog. In this study, we have analyzed the expression of ERbeta in formalin-fixed, paraffin-embedded tissue samples of nonaltered mammary gland, 30 malignant (six complex carcinoma, 12 simple carcinoma, three carcinosarcoma, and nine carcinoma or sarcoma in benign tumor), and five benign (one fibroadenoma, one complex papilloma, one complex adenoma, and two benign mixed tumors) mammary tumors of the dog by using a polyclonal ERbeta antibody and the avidin-biotin-peroxidase complex immunohistochemical technique. Our results show that high numbers of normal ductal and acinar epithelium and approximately one third of canine mammary tumors express ERbeta. This expression was higher in benign than in malignant tumors. Furthermore, expression was higher in complex and mixed histologic subtypes of malignant tumors when compared with simple subtypes.