Barbara Brunetti

E-Cadherin and β-catenin Reduction Influence Invasion but not Proliferation and Survival in Canine Malignant Mammary Tumors

E-Cadherin and β-catenin are known for their role in tumor invasion, but both proteins also exert an influence on tumor proliferation. This study, performed on canine mammary tumors, aimed to analyze the influence of E-cadherin (E-cad) and β-catenin (β-cat), immunohistochemically assessed singly and in combination (E-cad/β-cat), on survival and their relationship with several proliferation indices (AgNOR index, MIB1 index, mitotic index). Immunohistochemistry was carried out on 60 formalin-fixed, paraffin wax-embedded specimens of canine mammary malignancies. The labeling was defined as preserved when prevalent on cell membranes of more than 75% of cells and reduced in other forms of expression (i.e., membranous less than 75%, cytoplasmic, and negative). E-cad, β-cat, and E-cad/β-cat were preserved respectively in 22, 12, and 11 out of 60 cases. Immunohistochemical expression of the two proteins in the same tumors was significantly correlated (P = 0.0001; R = 0.57). Survival analysis revealed no difference in outcome comparing the preserved versus reduced cases (E-cad, P = 0.31; β-cat, P = 0.29; E-cad/β-cat P = 0.36). Grouping cases for histologic invasiveness, the expression of E-cad or β-cat and E-cad/β-cat showed a progressive reduction that paralleled an increase in invasiveness from noninfiltrating to stage-II tumors (E-cad, P < 0.001; β-cat, P < 0.05; E-cad/β-cat, P < 0.05). No significant difference was obtained comparing mitotic index, MIB 1 index, and AgNOR index by analysis of variance between the cases grouped for preserved or reduced E-cad, β-cat, and E-cad/β-cat variables. In conclusion, reduced expression of E-cad, β-cat, or E-cad/β-cat was significantly associated with the progression from noninfiltrating to highly infiltrating tumors but not with proliferation or survival.

E-Cadherin Immunoreactivity in Canine Mammary Tumors

The reduction or loss of E-cadherin (E-cad), a calcium-dependent epithelial cell adhesion molecule, has been associated with tumor dedifferentiation and invasiveness. The immunohistochemical pattern of E-cad expression was evaluated in formalin-fixed and paraffin-embedded sections of 6 normal mammary glands, 3 dysplasias, 12 benign tumors (8 benign mixed tumors, 4 adenomas), and 60 malignant tumors (12 stage 0, 29 stage I, 19 stage II) of the canine mammary gland. E-cadherin expression was classified as membranous, when on cell–cell boundaries, or as cytoplasmic, when in the form of a diffuse cytoplasmic staining. In addition, the percentage of E-cad–positive epithelial neoplastic cells was graded by a semiquantitative method, categorizing cases into a reduced (or -) type group, when showing less than 25% positivity, a reduced (or +/-) type group, when showing 25–75% positivity, and a preserved (or +) type group, when more than 75% positive cells were present. In the normal mammary gland, E-cad expression was evident in epithelial luminal cells. A stronger positivity was revealed in ductular than in alveolar luminal cells. The myoepithelial cells showed inconsistent, weak cytoplasmic positivity in the normal gland as well as in mammary tumors. In normal glands and benign and malignant noninvasive tumors, E-cad expression was mainly membranous and preserved in most of the epithelial cells. In stage I tumors, both membranous (38%) and cytoplasmic (62%) positivity were well represented, as well as preserved type (55%) and reduced type (45%) tumors. All stage II malignant tumors showed the highest frequency of cytoplasmic positivity (79%) and reduced type (62%) tumors.

Molecular Phenotype in Mammary Tumours of Queens: Correlation between Primary Tumour and Lymph Node Metastasis

The molecular characterization of mammary tumours represents a new stage in the development of effective predictive models and targeted therapies. The aim of this study was to evaluate the relationship between the molecular phenotype of a primary feline mammary tumour and that of a related lymph node metastasis. Twenty-one mammary tumour samples and their lymph node metastases were selected and evaluated immunohistochemically for expression of oestrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 (c-erbB-2), cytokeratin 5/6, cytokeratin 14, cytokeratin 19 and protein 63. Mammary tumours were classified into five subtypes: luminal A, luminal B, c-erbB-2 overexpressing, basal-like and normal-like, based on an algorithm applied in both human and veterinary medicine. Concordance between the primary tumour and its lymph node metastasis was detected in 12 of 21 cases (57.1%). In the remaining nine cases (42.9%) there was discordance in the molecular profile at the two sites. Therefore, the tumour molecular profile must be evaluated in both sites in order to obtain definitive identification of the tumour profile (or profiles) and to plan an appropriate therapy.

Characterization of Interstitial Nephritis in Pigs with Naturally Occurring Postweaning Multisystemic Wasting Syndrome

Kidney samples with interstitial nephritis from 26 pigs affected by postweaning multisystemic wasting syndrome (PMWS) were selected. A histologic evaluation was carried out to describe the type of inflammation and its relationship with viral load, as assessed by in situ hybridization (ISH). Of 26 cases, 10 revealed a tubulointerstitial, lymphoplasmacytic nephritis, 11 an interstitial granulomatous nephritis, and 5 both types of inflammation (mixed type). In 4 cases of granulomatous inflammation, the pattern was not classically nodular, and a population of macrophages and lymphocytes was present (interstitial lymphohistiocytic nephritis). ISH confirmed the presence of porcine circovirus type 2 (PCV2) nucleic acid in all cases. The epithelium of the renal tubules was the most constantly ISH-positive structure. In tubulointerstitial nephritis, the higher the number of positive inflammatory cells, the more severe the inflammation. The ISH reaction was more heterogeneous and unpredictable in granulomatous nephritis, with some epithelioid and giant cells positive by ISH. To quantify macrophages distributed in the three patterns of nephritis, immunohistochemical methods using anti-major histocompatibility complex II (anti-MHC-II) and anti-lysozyme antibodies were undertaken, and semiquantitative evaluation was carried out. MHC-II was mainly expressed by lymphocytes in tubulointerstitial nephritis, but did not always stain macrophages in cases of granulomatous (including lymphohistiocytic) nephritis; the anti-lysozyme antibody revealed macrophages when present in tissues. The amount of PCV2 nucleic acid was not apparently associated with the pattern of inflammation (tubulointerstitial or granulomatous). PCV2 load seems to reflect the severity of the lymphoplasmacytic inflammation but not that of granulomatous and lympho histiocytic types.

Lymphatic vessels assessment in feline mammary tumours

BackgroundThe lymphatic vessels play a crucial role in a variety of human cancers since tumour cell lymphatic invasion significantly influences prognosis. It is not known if pre-existing lymphatics are enough for tumour dissemination or de novo development is necessary. VEGFR-3 is an angiogenetic mediator for both lymphatic and blood vessels during embryonic development, and only for lymphatics after birth. VEGF is a mediator of both vasculogenesis and angiogenesis, regulates the growth of lymphatics in various experimental models, and is produced in many solid tumours. CD44 mediates hyaluronic acid (HA)-dependent cell adhesion: besides promoting invasion, this interaction also supports neoangiogenesis that indirectly stimulates tumour cell proliferation. The expression of VEGF-C (Vascular Endothelial Growth Factor – C), its receptor VEGFR-3 and CD44, were studied on feline mammary samples to assess the importance of lymphangiogenesis and lymphangiotrophism in neoplasia.MethodsSamples were taken from six normal mammary glands (NMG), ten benign (BT) and 32 malignant (MT) tumours. Immunohistochemical laminin/VEGFR-3 double stain, VEGF-C and CD44 stains were applied to 4 μm-thick sections, and their expression evaluated in intratumoral/extratumoral and intramammary/extramammary fields.ResultsAll groups revealed a higher number of lymphatics in the extratumoral/extramammary areas. VEGF-C expression in the epithelium paralleled the number of positive vessels in the NMG, BT and MT, whereas VEGF-C higher expression was noted in the intratumoral fields only in infiltrating MT. CD44 score was lower in extratumoral than intratumoral fields in tumours and showed a significant increase in extramammary/extratumoral fields from NMG to MT. Pearson test showed a significant and inversely proportional correlation between CD44 expression and the number of lymphatic vessels with VEGFR-3 in malignant infiltrating tumours.ConclusionThe number of both VEGFR-3 positive and negative lymphatics in the extratumoral and extramammary stroma was significantly higher than intratumoral and intramammary fields respectively in the NMG, BT and MT. This suggests a scant biological importance of intratumoral lymphatics while their higher number is due to the concentration of existing vessels following compression of the extratumoral stroma in spite of a non demonstrable increase from NMG to MT. The tumour model employed provided no evidence of lymphangiogenesis, and metastasis in the regional lymph node develops following the spread through the pre-existing lymphatic network.

Quantitative Analysis of Telomerase in Feline Mammary Tissues

The purpose of this study was to validate immunohistochemistry (IHC) as an alternative to telomerase repeat amplification protocol (TRAP) analysis to detect telomerase activity. TRAP–enzyme-linked immunosorbent assay (ELISA) reactivity was compared with telomerase reverse transcription (TERT) IHC staining in 22 feline mammary tissues (6 normal mammary glands, 2 dysplastic mammary glands, 1 fibroadenoma, and 13 malignant neoplasms [6 solid mammary carcinomas, 2 squamous-cell carcinomas, 4 tubulopapillary mammary carcinomas, and 1 mammary carcinosarcoma]). TERT IHC staining revealed enzymatic expression in nuclear, nucleolar, cytoplasmic, and combined nuclear and nucleolar staining patterns that were separately quantified by image analysis and expressed as the absolute number (average) of positive cells or percentage of positive cells with respect to overall cellularity. With TERT IHC staining, the absolute number and percentage of cells with positive nuclei and nucleoli within the same cell were the variables with the greatest discrimination between benign and malignant mammary lesions (analysis of variance [ANOVA], average P < 0.0001; percentage P < 0.001). For TRAP-ELISA–positive versus TRAP-ELISA–negative tissues, a positive test result provided greater differentiation between malignant versus benign mammary lesions (ANOVA, average P = 0.00038; percentage P = 0.0022). The same IHC pattern of expression showed a proportional and significant (average P = 0.004; percentage P = 0.002) but low (average R = 0.60; percentage R = 0.63) correlation with TRAP-ELISA by the Pearson test. The correlation coefficients obtained show that IHC and TRAP cannot be considered interchangeable because the 2 methods are more complementary than exclusive.

Mammary Mucinous Carcinoma in the Cat

Mucinous carcinoma of the mammary gland is a rare tumor characterized by excessive mucin production. In human and canine pathology, the diagnosis of mucinous carcinoma is based on the demonstration of an epithelial phenotype of mucus-producing cells and periodic acid-Schiff (PAS)-diastase positivity of the mucin. The histologic and immunohistologic characteristics of feline mucinous mammary carcinoma were examined. Of 656 cases of feline mammary neoplasms and dysplasias, 3.2% were found to be mucin-producing tumors. Cytokeratin 19 (16 cases positive, 4 heterogenous, and 1 negative) and vimentin (15 cases positive, 2 heterogenous, and 4 negative) expression were examined, and the mucin produced was alcian blue positive. PAS-diastase staining was variable (38.1%). Based on these findings, mucinous mammary carcinoma in the cat varies significantly from the human and canine varieties and alcian blue is the prominent stain in the diagnosis of feline mucinous carcinoma.

Evaluation of Telomerase in Canine Mammary Tissues by Immunohistochemical Analysis and a Polymerase Chain Reaction-Based Enzyme-Linked Immunosorbent Assay

The enzyme telomerase is considered a potential marker for neoplastic tissue and is used as a diagnostic and prognostic tool in clinical medicine and therapeutics. For this reason, the possible role of telomerase activation in the process of malignant transformation is currently the subject of intense research efforts. The focus of the study reported here was to detect telomerase in 37 canine mammary samples, by comparing two methods: immunohistochemical (IHC) analysis for detecting the catalytic subunit of the enzyme, telomerase reverse transcriptase (TERT), and the telomeric repeat amplification protocol–enzyme-linked immunosorbent assay (TRAP-ELISA), a polymerase chain reaction (PCR)-based technique that uses a colorimetric detection method. Using the TRAP-ELISA, samples were considered positive when they yielded a difference of at least 0.2 absorbance units between the readings at 450 nm versus 690 nm wavelength. On the basis of this criterion, 18 negative and 19 positive cases were obtained. Specific immunohistochemical staining was observed mainly in the nucleoli, to a lesser extent in the nuclei, and rarely in the cytoplasm of epithelial cells. A sample was considered positive when at least 10% of the epithelial cells had specific staining. The Pearson correlation between the TRAP-ELISA and IHC results was significant only when IHC nucleolar (r = 0.53, P < 0.01) or nuclear (r = 0.36, P < 0.05) staining or their combination (r = 0.58, P < 0.01) was considered. Thus, IHC staining of nucleoli and nuclei can be considered as an alternative method to the TRAP-ELISA. The detection of telomerase in normal mammary gland and fibrocystic mastopathy using both methods does not support the idea that telomerase may be used as a specific marker of mammary neoplasia in dogs.

Chronic rhinitis due to Streptococcus equi subspecies zooepidemicus in a dog.

PRIMARY bacterial rhinitis is rare in dogs; it is usually secondary to nasal trauma, inhalation of an irritant, neoplasia, or viral, fungal or parasitic infections. Streptococcus equi subspecies zooepidemicus ( S zooepidemicus ), a common cause of respiratory infections in horses, has been

Histochemical and immunohistochemical characterization of canine mammary mucinous carcinoma.

Mucinous carcinoma is a rare mammary tumour, characterized by intracellular and extracellular mucin. It is still uncertain whether the origin of the mucin is epithelial, myoepithelial or fibroblastic. Eleven canine cases originally classified as mucinous carcinomas were reassessed and compared with myoepithelial nests of mixed tumours. All samples were examined (1) histochemically by the periodic acid-Schiff (PAS) and PAS-diastase methods, and with alcian blue (pH 2.5 and pH 1.0), mucicarmine and Grimelius silver stain, and (2) immunohistochemically for cytokeratin 19, vimentin, alpha-actin and chromogranin A. This examination revealed that only five of the 11 tumours were genuine mucinous carcinomas. In these five tumours the mucus-secreting cells showed cytoplasmic cytokeratin 19 positivity; the mucus showed PAS-diastase and mucicarmine positivity, and alcianophilia which was stronger at pH 2.5 than at 1.0. The remaining six cases were re-classified as mixed tumours because both mucus and mucus-producing cells shared the following similarities with myoepithelial nests of mixed tumours: vimentin and alpha-actin cytoplasmic positivity, PAS negativity, alcianophilia both at pH 2.5 and 1.0, and mucicarmine positivity.