CHARACTERIZATION OF THE PROLIFERATION STATE IN CANINE MAMMARY-TUMORS BY THE STANDARDIZED AGNOR METHOD WITH POSTFIXATION AND IMMUNOHISTOLOGIC DETECTION OF KI-67 AND PCNA

Paraffin-embedded tissue sections of 96 surgically removed mammary tum ors from female dogs were analyzed for their proliferation state using three different methods. The AgNOR method, originally developed by Pl oton and coworkers in 1986,(34) modified and standardized by the AgNOR committee, is an easy, inexpensive silver-staining procedure used to detennine cell proliferation and prognosis of various tumors. Due to t he standardized staining protocol of the AgNOR method and a postfixati on step, results obtained were of excellent quality for image-analysis processing. The growth fraction was evaluated by counting of immunohi stologically positive-stained cells for Ki-67 or proliferating-cell nu clear antigen (PCNA). The values determined were, in general, lower wi th Ki-67 (MIB1) than with PCNA (PC10). Nevertheless, the labeling indi ces of these antigens correlated significantly (P <0.001). Though the differences of the means between the tumor groups according to the cla ssification system of the World Health Organization were significant f or all three investigated methods (P <0.001), there was a considerable overlap between the tumor groups concerning all investigated paramete rs. An exploratory data analysis (multivariate analysis) was performed to evaluate the prognostic relevance of the three methods including f urther anamnestic, clinical, gross, and histopathologic variables. Bes ides the histopathologic diagnosis (survival P <0.001; survival time P <0.05; reappearance of tumor growth P <0.05). only the PCNA-labeling index (time until reappearance of tumor growth P <0.001) was of progno stic significance.