Materials and Methods

Animals and Samples. Serum and tissue samples of 30 animals presented at Veterinary Teaching Hospital of Veterinary School (Madrid), with at least one mammary tumor, were prospectively collected. The animals were physically and radiographically examined. Surgical excision was the treatment ofchoice except in IMC cases, in which surgery is not the first choice recommended. Samples were obtained by "tru-cut" biopsy or necropsy. For this study, samples were divided in two adjacent fragments, one was fixed in 10% neutral formalin (for histological and immunohistochemical study), and the other was frozen and stored for hormonal detection. Five control animals without history of any disease were included , serum and samples oftwo different mammary glands were obtained. To determine the esrus cycle at sampling, vaginal smears were performed.

Hormonal Study. From 35 female dogs, a total of eighty-six mammary samples were collected: 10 normal mammary gland (NMG), 21 dysplasias (DYSP), 26 benign (BMT), 22 malignant (MMT), and 7 IMC.

Plasma and Homogenate IGF-I Ensymeimmunoassay (EIA). IGF-I levels both in plasma and tissue homogenates were determined by a commercial assay kit (Non-extraction IGF-I ELISA, DSL-10-2800), validated for species and tissues.

Plasma and Homogenate DHEA, Androstenedione, and Testosterone EIA.

Plasma and tumor homogenate DHEA, androstenedione and testosterone concentrations were assayed by amplified EIA previously validated for this species and tissues (12).

Histopathology. Immunohistochemistry of IGF-1R and AR. The samples for histopathology and immunohistochemistry were fixed in 10% buffered formalin; paraffin embedded and cut in sections, following routine methods.

Histopathological diagnosis was done on H&E sections following the WHO's classification for canine mammary tumors and dysplasias .

For immunohistochemistry, a selection of each tissue group was used: NMG (n = 5), BMT and DYSP (n = 10), MMT-non-CI (n = 20), MMTCI (n = 20) and 13 achieved CI cases were also included. Immunohistochemistry was performed on deparaffined sections using the streptavidin-biotin-complex peroxidase method after a high temperature unmasking protocol. The primary antibodies used were a mouse monoclonal antibody anti-IGF-1Ra subunit (clone 24-31, Neomarkers Int.; dilution 1/50; incubation overnight at 4°C), and a polyclonal rabbit anti-AR (clon 2F12, Novocastra dilution 1/10; incubation overnight at 4°C). Known positive and negative control samples were used. The intensity of the staining was evaluated in all sections luated by two observers simultaneuously according to the following scale: Negative = 0, Low = +, Moderate = ++, and Intense = +++.

Statistical Analysis. The Biomedical Data Program (BMDP), Statistical Software Inc. (Los Angeles, CA, USA), was used for statistical analysis. In all statistical comparisons, P< 0.05 was accepted as denoting significant differences.

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