Rosario Preziosi

Prognostic Value of Histologic Stage and Proliferative Activity in Canine Malignant Mammary Tumors

The aim of this study was to investigate the correlation between the histologic invasiveness (histologic stage) and various cell proliferation activity assays (quantity of argyrophil proteins associated with nucleolar organizer regions [AgNORs], mitotic activity, MIB1 [Ki67] immunohistochemical detection) for predicting the biologic behavior of malignant canine mammary tumors. Sixty specimens from malignant canine mammary tumors with no distant metastases (M0) at surgery were selected, and follow-up data were collected over a 2-year period. The histologic invasiveness was graded by histologic stage (stage 0 = tumors without stromal invasion; stage I = tumors with stromal invasion; stage II = tumors with neoplastic emboli in vessels), and the proliferative indices were expressed as MIB1 index (the percentage of nuclear area immunohistochemically stained by MIB1 antibody), mitotic index (the number of mitoses per 1,000 neoplastic cells), and AgNOR index (the ratio between mean AgNOR area of tumor cells and the mean AgNOR area of fibroblasts/lymphocytes). The measures of proliferative activity were compared among groups with different histologic stages, and the influence of different prognostic variables (histologic stage, AgNOR index, mitotic index, MIB1 index) on survival time was evaluated. A significant difference in the proliferation patterns was recorded between the different histologic stages for the mitotic index (P = 0.0006) and MIB1 index (0.0013). Among the different parameters considered, histologic stage (P < 0.05), AgNOR index (P = 0.0291), and MIB1 index (P = 0.014) revealed a significant association with prognosis in univariate analysis. AgNOR index for 1-year survival and histologic stage for 2-year survival were the most significant parameters influencing survival, as determined by multiple nonlinear logistic regression.

Detection of proliferating cell nuclear antigen (PCNA) in canine and feline mammary tumours

Quantitation of immunohistochemical staining of the proliferating cell nuclear antigen (PCNA, clone PC10) by image analysis was performed on benign and malignant mammary tumours of dogs and cats. Scoring of the slides was carried out by image analysis to assess the percentage of labelled nuclei (expressed as a ratio of areas). Either the strongly labelled nuclei (SP-PCNA index), or all of the stained nuclei (TP-PCNA index) were counted as positive to determine the growth fraction and its correlation with the histopathological classification and nuclear grade (degree of nuclear differentiation, considered a morphological correlate of tumour aggressiveness). A significant difference in the values of PCNA indices was seen between benign and malignant growths (P < 0.0001, dog; P < 0.05, cat). Neither of the PCNA indices showed correlation with nuclear grade in dogs (P = 0.14 for SP-PCNA index and P = 0.31 for TP-PCNA index) or cats (P = 0.09 for SP-PCNA index and P = 0.07 for TP-PCNA index). A significant difference in the number of mitoses, expressed as mitotic index, was seen between benign and malignant growths in the dog (P < 0.01) but not in the cat (P = 0.078). Good correlation of mitotic index with nuclear grade was revealed in canine malignant growths (P < 0.05), but in feline malignant tumours such correlation (P < 0.05) was shown only when the values of intermediate plus typical forms were compared with the data for atypical forms. It is concluded that quantitation of PCNA-positive nuclear area by image analysis provides an objective method for assessing proliferative activity in benign and malignant mammary tumours of dogs and cats.

C-kit Gene Product (CD117) Immunoreactivity in Canine and Feline Paraffin Sections

CD117 is a transmembrane tyrosine kinase growth factor receptor expressed by a variety of normal human cell types, including germ cells, immature myeloid cells, and mast cells. To evaluate the pattern of CD117 expression in dogs and cats, we applied a polyclonal antibody on paraffin sections from 44 samples of normal tissues and 104 tumors. In both species, strong immunoreactivity was observed in mast cells, interstitial cells of Cajal, and in mast cell tumors. Among gastrointestinal mesenchymal neoplasms, tissues from five dogs and one cat revealed strong CD117 expression, enabling us to identify them as gastrointestinal stromal tumors (GISTs).

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.

Multivariate Survival Analysis of Histological Parameters and Clinical Presentation in Canine Cutaneous Mast Cell Tumours

The Patnaik histological grade is a good method for the prediction of long-term mast cell tumour behaviour but it is influenced by subjective inter-observer variations and intratumoral heterogeneity. The present study evaluated each of the histopathological parameters used to formulate Patnaik’s grade in terms of prognosis and tested whether they have a different prognostic sensitivity, thereby disclosing which could be considered more useful in the prediction of tumour recurrence and patient survival. Clinical presentation (single or multiple tumours) was also considered as possible prognostic factor. The results demonstrated that individual histological criteria together with multiple presentation may be of value in predicting the outcome of mast cell tumours. Among these, invasiveness (β1.85; standard error 1.15) and the number of mitotic figures (β3.01; standard error 1.18) showed high prognostic significance (Cox proportional hazard regression for censored data ; chi-squared = 15.52, degree of freedom = 6, p = 0.016) and could serve as reliable prognostic indicators avoiding more subjective parameters such as cellular differentiation, nuclear morphology and tumoural pattern.

Proliferative activity assessed by anti-PCNA and Ki67 monoclonal antibodies in canine testicular tumours.

The recent availability of monoclonal antibodies raised against cell cycle nuclear antigens makes possible, by means of immunohistochemical techniques, an easy and quick method of evaluating tumour kinetic activity, in addition to older methods such as measurement of the mitotic index. Some of these antibodies can be used on formalin-fixed paraffin wax-embedded samples, thus allowing the use of archival material. In the present study the proliferative activity of testicular tumours of the dog (seminomas and Sertoli and Leydig cell tumours) was investigated with two monoclonal antibodies to proliferating cell nuclear antigen (PCNA) clone PC10, and Ki67 clone MIB1. The former recognizes a formalin-resistant epitope of PCNA, and MIB1 the same antigen as Ki67 in formalin-fixed, paraffin wax-embedded sections after incubation in a microwave oven. Three parameters of proliferative activity were considered: PCNA and Ki67 indices (percentage of nuclear area positive to PCNA and to Ki67), and mitotic index (number of mitoses per 1000 cells). The PCNA index and Ki67 index revealed a good correlation in linear regression analysis (P < 0.001) as did the mitotic index (P < 0.01). None of the parameters considered revealed a significant difference in proliferative activity of the three types of tumour (P > 0.05-Spearman test), but in both seminomas and Sertoli cell tumours the progression from tubular to diffuse pattern paralleled an increase in growth fraction. It is interesting that some seminomas of the diffuse type, often considered on histological grounds to be the most malignant, showed the highest values of the above-mentioned parameters.

Expression of the KIT protein (CD117) in primary cutaneous mast cell tumors of the dog

Thirty-one canine cutaneous masses, diagnosed as mast cell tumors (MCT) by histopathologic analysis, were used to evaluate the immunohistochemical pattern of expression of KIT protein (CD117), a type III tyrosine kinase protein involved in mast cell growth and differentiation. Lesions were graded as I (well differentiated), II (intermediate differentiation), or III (poorly differentiated) according to the following morphologic features: invasiveness, cellularity and cellular morphology, mitotic index, and stromal reaction. Immunohistochemical KIT expression was compared with histologic grade and some histomorphologic features (cell differentiation and nuclear grade) evaluated separately. A possible predictive role of biologic behavior in MCTs for KIT expression was also investigated. Immunohistochemical analysis revealed three different patterns of KIT expression: a cytoplasmic diffuse pattern, a membranous pattern with immunostaining located on the cell surface, and a cytoplasmic perinuclear pattern, where KIT expression was detected in the cytoplasm of the neoplastic mast cells, close to the nucleus. Statistical analysis showed a close relationship between different KIT immunohistochemical patterns and histologic grade (P < 0.00000), cell differentiation (P < 0.00000), and nuclear grade (P < 0.0024). According to Kaplan-Meier–estimated survival curves compared by survival analysis, KIT expression was significantly associated with survival time (P = 0.037) but not cancer-free interval (P = 0.50). Similar to other well-known histomorphological features, KIT expression is a useful parameter of malignancy in cutaneous MCTs. KIT expression also predicted the biological behavior of the tumors in this study.

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.

Multiparametric survival analysis of histological stage and proliferative activity in feline mammary carcinomas

Mammary tumours are among the most frequent malignant neoplasms in the cat and determination of prognosis on histological grounds alone can be unsatisfactory because it does not always correspond to the clinical behaviour of the neoplastic disease. The aim of this two-year post-mastectomy survival study is to relate the histological stage or invasiveness (the most commonly used histological parameter to grade malignancy) to several parameters assessing the proliferative activity-mitotic index, MIB1 index, and AgNOR index. Invasiveness was graded as local and vascular invasion whilst values of the parameters expressing proliferative activity, all quantified by image analysis, have been classified into low and high proliferative activity groups according to their median values, (0.719 for mitotic index, 12.11 for MIB1 index, and 3.19 for AgNOR index). For each group, mean survival (months+/-SD) was calculated. Histological stage (local invasion 21.83+/-7.83 months, blood vessels and/or lymphatics invasion 13.38+/-8.99,P<0.01), mitotic index (low 22.43+/-88.78, high 12.37+/-7.49,P<0.001), and AgNOR index (low 21.86+/-10.68, high 13.82+/-7.11,P<0.05) revealed a significant association with survival in univariate analysis and had an independent prognostic value in multiparametric survival test (P<0.001).

Hypertrophy of intestinal smooth muscle in cats

Pathological findings of four cats with severe and diffuse smooth muscle hypertrophy of the small intestine (MHSI) are reported and compared to those of five cats with segmental MHSI secondary to neoplastic obstruction and four controls. Histology demonstrated a constant association between idiopathic MHSI and submucosal fibrosis and chronic lymphoplasmacytic enteritis. Morphometry (gut diameter, thickness and area of muscular layers, number and density of smooth muscle nuclei) and MIB-1-immunolabelling showed that the thickness increase was mostly due to hypertrophy, but hyperplasia was also evident. Microbiology from ileal content samples was performed in two cats with primary MHSI, and Campylobacter spp. were isolated, which were also demonstrated by immunohistochemistry and ultrastructure. The association of chronic enteritis with idiopathic MHSI suggests that factors released in intestinal inflammation may also act as hypertrophy stimuli for smooth muscle cells.