Tissue culture of feline normal mammary gland

To study the growth and morphology of feline mammary epithelial cells in vitro, normal feline mammary tissues as well as cells collected from feline milk were cultured and examined using phase-contrast microscopy and immunohistochemistry. The method employed was a modification of Hiratsuka et al., and was found to be useful for the culture of dissociated feline mammary tissue. Small polygonal cells and large spindle-shaped cells that were positively stained with anti-keratin and anti-actin antibodies formed colonies with a pavement-like structure in culture of mid-pregnant mammary gland. Cultures of involuting mammary gland were composed mainly of small spindle-shaped cells, which were negative for keratin immunostaining and thought to be fibroblasts. Myofibroblasts, which participate in feline mammary carcinogenesis, and show keratin negative, actin positive, were not encountered in the present culture system of normal mammary gland. The cells from milk formed small colonies, but did not grow to reach confluent monolayer.     

Nuclear cytomorphometry in feline mammary gland epithelial tumours

Stained cytological specimens from 35 feline mammary gland epithelial tumours (4 adenomas, 11 tubulopapillary carcinomas 13 solid carcinomas and 7 cribriform carcinomas) were analysed by computer-assisted nuclear morphometry. In each case, the nuclei of at least 100 neoplastic cells were measured, and the mean nuclear area (MNA), mean nuclear perimeter (MNP), mean nuclear diameter (MND) and nuclear roundness (NR) were calculated. The study aimed to evaluate (1) the possibility of using nuclear cytomorphometry as an auxiliary diagnostic method to differentiate between benign and malignant feline mammary gland epithelial tumours, and (2) the prognostic value of nuclear morphometry in feline mammary carcinomas. The results indicated that MNA, MNP, MND and NR could be a useful adjunct in diagnosis but are not reliable prognostic indicators for feline mammary gland carcinomas.     

Ultrasonographic findings of feline cholangitis

Cholangitis is a common inflammatory disorder of the biliary system in cats. There are two major forms based on the predominate type of inflammatory cell infiltrates: lymphocytic or neutrophilic. Ultrasound is a common imaging modality used in these patients. This retrospective study evaluated the ultrasound examinations of 26 cats with a histologic diagnosis of cholangitis. Most cats with cholangitis had sonographically normal liver size, echogenicity, and normal biliary systems. Statistically significant sonographic changes for cats with cholangitis included hyperechoic liver parenchyma, hyperechoic gallbladder contents, and increased pancreatic size. No statistically significant changes were noted to distinguish lymphocytic and neutrophilic forms of cholangitis. Cats with the sonographic features of diffuse liver hyperechogenicity, gallbladder contents and enlarged pancreas may suggest cholangitis.     

Ultrasonographic study of the feline sciatic nerve

This study was conducted to describe the ultrasonographic appearance and technique for the evaluation of sciatic nerve (ScN) in cats. An anatomical study was carried out using six feline cadavers to determine anatomic landmarks. An ultrasonographic 'in vitro' study was performed using eight pelvic limbs from four fresh feline cadavers to assess the ScN in three regions (glutea, femoralis and popliteal) using a 4-13MHz linear transducer. Five healthy adult experimental cats were employed for an 'in vivo' ultrasonographic study of the ScN using the same protocol described for the 'in vitro' study. The ultrasonographic images of the ScN were well correlated with those obtained in the anatomical study. The ScN was easily identified in all the approaches. The ScN was observed as a hypoechoic structure with internal echoes, outlined by hyperechoic lines. This study shows the usefulness of ultrasound to evaluate the entire pathology of the ScN in the cat.     

Ultrasonographic appearance of edema caused by injections in the mammary gland attachments of dairy cows

OBJECTIVE: To determine the ultrasonographic appearance and detectability of edema induced by SC injection of mild silver protein suspension in the mammary gland attachments of dairy cows. DESIGN: Prospective study. ANIMALS: 6 lactating cows. PROCEDURE: In each cow, the number of quarters that received injections was randomly assigned. A mild silver protein susoension was injected SC into cranial and caudal mammary gland attachment sites. The number of injections and volume injected were determined on the basis of the appearance of the mammary gland and the desired subjective visual effect. Seventeen sites were chosen for injection and 7 sites did not receive injections. Ultrasonographic images were obtained 1 day prior and 6 days after injections were started. Cows received injections 1, 3, and 5 days after initial sonography. The sonographer was unaware of which sites received injections. RESULTS: Ultrasonography revealed alternating hypoechoic and hyperechoic bands at injection sites. Certain injections caused the intimal surface of the subcutaneous abdominal vein to develop a corrugated appearance. All injection sites were correctly identified ultrasonographically (100% sensitivity, 100% specificity) with a positive and negative predictive value of 1.0. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that mild silver protein suspension injected SC to enhance the appearance of the mammary glands of dairy cows can be readily detected by ultrasonography. Detection of injection sites should be made on the basis of the distribution and ultrasonographic appearance of edema.     

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.     

The expression of sialyl Lewis X in canine and feline mammary gland tumors

The expression of sialyl Lewis X (sLe(x)) in 93 canine and 15 feline mammary gland tumors (MGT) obtained by surgical resection at Veterinary Medical Center, the University of Tokyo was examined by immunohistochemistry. Their clinicopathological features and prognosis were also reviewed. Approximately 60% of MGT tissues showed sLe(x) positive expressions, while all normal mammary gland tissues were negative. However, its expression was not correlated with clinicopathological features and prognosis significantly. This study suggests that sLe(x) may be a tumor-associated antigen in canine and feline MGTs.     

Retrospective evaluation of adjunctive doxorubicin for the treatment of feline mammary gland adenocarcinoma: 67 cases

Medical records for 67 cats with histologically confirmed mammary gland adenocarcinomas treated with adjunctive doxorubicin from June 1994 through December 2002 were reviewed. Data were examined to evaluate factors influencing disease-free interval (DFI) and survival time. The Kaplan-Meier median survival time of cats that received surgery and doxorubicin was 448 days. The Kaplan-Meier median DFI was 255 days. Significant univariate prognostic factors for DFI included histological subtype, completion of initial chemotherapy, development of metastatic disease, and location of metastatic disease. Significant univariate prognostic factors for survival included tumor volume, the development of metastatic disease, and location of metastatic disease.     

Ultrastructure of feline mammary hypertrophy

The ultrastructure of feline mammary hypertrophy was studied in a five-month-old female which had aborted recently, a ten-year-old female which was one month postestrus, and a four-year-old progestin-treated neutered male. Morphologic comparisons were made to normal mammary tissue from a one-year-old female cat. Hypertrophied mammary tissue had the same cell types and spatial relationships as did the normal gland. Major differences included a more highly developed duct system composed of metabolically active cells which often were arranged in multiple cell layers, and periductular stroma with increased fibroblasts and vascularization. Hypertrophied epithelial cells were characterized generally by smooth-contoured nuclear membranes, more evenly dispersed heterochromatin, prominent nucleoli, increased polyribosomes, and elongated mitochondria. Secretory activity was developed significantly only in the cat that had aborted recently. Modifications in myoepithelial cells included: more evenly dispersed nuclear heterochromatin, thicker bundles of cytoplasmic filaments, more straight plasma membranes along the basal lamina, and elongated hemidesmosomes. Multilayering of the basal lamina was accentuated. Stromal fibroblasts had nuclear heterochromatin distributed similarly to that of epithelial and myoepithelial cells, and increased rough endoplasmic reticulum. Myoepithelial cells did not contribute to the increased stromal cellularity. No significant ultrastructural differences were noted between mammary hypertrophy in young, old, and progestin-treated cats.     

Activation of AKT in feline mammary carcinoma: a new prognostic factor for feline mammary tumours

The PI3K/AKT/PTEN pathway is involved in the pathogenesis of several human cancers. This study investigated the biological and prognostic value of PI3K/AKT/PTEN pathway dysregulation in feline mammary tumours. Expression of p-AKT, HER2, PTEN and steroid receptors was assessed by immunohistochemistry (IHC) in 27 malignant and 12 benign mammary tumours from 39 female cats followed up over a 24-month period. Feline mammary carcinoma (FMC) cell lines were analyzed by Western blot and the feline AKT gene sequence was characterized. p-AKT expression statistically correlated with tumour malignancy, histological dedifferentiation and clinical recurrence. The animals with tumours expressing p-AKT had a shorter disease-free period than those with p-AKT-negative tumours. AKT activation was associated with HER2 expression and PTEN down-regulation, as occurs in human breast cancer, and feline AKT sequencing showed high homology with the human AKT gene. No AKT activation was observed in relation to either oestrogen receptor α (ERα) or progesterone receptor expression. Taken together, these data offer an explanation for AKT signalling and its role in FMC pathogenesis and prognosis, shedding new light on similarities between feline mammary tumours and hormone-independent breast cancer.     

Progesterone receptors in feline mammary adenocarcinomas

Estradiol and progesterone receptors were measured in tumor cytosols from 3 intact and 4 neutered female cats with spontaneously occurring mammary adenocarcinomas. Serum from 2 of the intact cats which had been in estrus 4 and 4 to 6 weeks before tumor excision contained progesterone concentrations of 16.2 and 2.2 ng/ml, respectively; serum progesterone in the other cats was less than 2 ng/ml. Estradiol receptors were not detected in any cytosols. Progesterone receptors were detected in all of the cytosols, in concentrations ranging from 4.0 to 11.7 (mean = 7.2) fmol/mg of protein. Scatchard plot analysis of tumor cytosol from an 8th cat with mammary adenocarcinoma revealed presence of high affinity progesterone binding with a dissociation constant (Kd) of 3.47 nM. Tumor receptor content could not be correlated with stage of the estrous cycle nor with whether the cat was intact or neutered.     

Prognostic factors for feline mammary tumors

The case records of 100 cats with malignant mammary tumors were reviewed. All cats were staged clinically according to the staging system of the World Health Organization. The following information was obtained from the medical records: age at time of diagnosis, breed, tumor size, date of surgery, type of surgical procedure performed, histologic type of tumor, disease-free interval, survival time, and cause of death. Factors of no prognostic value were age (less than or equal to 10 years vs greater than 10 years) and breed. Tumor size was the most significant prognostic factor (P less than 0.0001). Cats with small tumors (1 cm3 to 8 cm3) had the best prognosis. The type of surgery, conservative vs radical, was significantly (P less than 0.01) related to disease-free interval, but was of no significance in prolonging survival time.     

The expression of carbonic anhydrase in canine mammary gland and mammary gland tumor

Carbonic anhydrase (CA), a metallo-enzyme containing zinc, broadly distributes in mammalian tissues and participates in physiological regulation such as respiration, acid-base balance, ion transport, bone resorption, as well as the development of tumor by the reversible hydration of carbon dioxide. However the expression of CA in the tissue of mammary gland tumor was not documented. In this study we examine the histolocalization and gene expression of CA in both normal canine mammary gland tissue and mammary gland tumor by histochemical examination, and RT-PCR. Four mRNA expression of CA isoenzymes, such as CA II, IV, VI and IX were found under RT-PCR analysis and different band patterns were found between normal canine mammary tissue and canine mammary gland tumor tissue. CA II, IV, VI and IX gene mRNA expression were found in the normal mammary gland tissue, indicating CA II, IV, VI and IX are likely to be the essential enzymes to maintain the normal physiological condition of canine mammary gland tissue cells. However the expression of CA IV was not found in the tissue of malignant mammary gland tumor that may become the marker for the prognostic recognition of canine mammary gland tumor.     

Canine mammary gland tumors

An understanding of the known biologic facts of this disease and methodical evaluation of the individual patient are necessary prerequisites for outlining rational courses of therapy for dogs afflicted with mammary tumors. Because of the common occurrence of multiple tumors arising at various stages of development simultaneously and the heterogeneity of histology of the complex (mixed tissue types) tumors, presurgical biopsy is recommended only in cases in which mast cell tumor or anaplastic carcinoma is suspected. Although investigative work is being performed regarding the efficacy of chemotherapy, radiation therapy, and immunotherapy, surgery still remains the mainstay in treatment of this condition. No one surgical procedure fits the needs of all patients, although it seems logical to remove as much breast tissue as is reasonable in each circumstance owing to the multicentric nature of the disease. Ovariohysterectomy has not been demonstrated to be of value in treatment of dogs with mammary tumors, but it is a markedly effective method of preventing mammary tumors if it is performed before puberty; it is moderately effective if performed before the dog is 21/2 years of age.     

Immunohistochemical analysis of estrogen receptors in feline mammary gland benign and malignant lesions: comparison with biochemical assay

Estrogen receptors (ER) were determined by both the biochemical dextran-coated charcoal (DCC-ER) and the immunohistochemical Avidin biotin-peroxidase complex (IHC-ER) methods in proliferative mammary lesions collected from 37 cats: 20 malignant tumors without metastasis at first presentation, seven malignant tumors with lung and/or lymph node metastases and 10 benign tumors and dysplasias. Total number of samples analyzed by both methods was 44. The DCC-ER method was applied to frozen tissue samples and the IHC-ER method was applied to neutral buffered formalin-fixed, paraffin wax-embedded tissue samples by using the NCL-6F11 monoclonal antibody. Biochemically, 21 (47.7%) cases had equal or more than 5 fmol/mg of protein (standard positivity threshold). Immunohistochemically, 11 (25%) cases were scored positive, the percentage of positive nuclei being statistically linked to the intensity of immunostaining. Normal mammary gland tissue (13 cases) and/or dysplastic areas (5 cases) found in the surroundings of the main lesion were IHC-ER positive in 76.9% and 40% of the cases, respectively. Concordance between DCC-ER and IHC-DCC was 72.7% and the results of the DCC and the IHC-ER methods were significantly correlated (P < 0.05) by the chi2 test. Specificity (true negatives) and sensitivity (true positives) of the ICH-ER method were 95.6% and 47.6%, respectively. One out of eleven DCC-ER positive and IHC-ER negative discordant cases (9.09%) was a DCC-ER false positive, because the surrounding normal mammary gland tissue was IHC-ER positive. The remaining 10 cases had ER content values equal or lower than 23 fmol/mg of protein, a figure that could represent the sensitivity threshold of the immunohistochemical method employed.