Wojciech Łopuszyński
Diagnostic and prognostic values of selected histopathological parameters and immunohistochemical indicators of proliferation and apoptosis in mammary gland tumours in female dogs
Mammary carcinomas are the most frequent neoplasms diagnosed in female dogs, and the main treatment is surgery. However, the high rate of recurrence and metastatic events after surgical treatment implies the necessity to look for accessory methods to enable the selection of those cases with an increased risk of early neoplastic progression. In dogs, the variability of histologic types and the especially diversified microscopic picture of mammary tumours are the main cause of the numerous discrepancies concerning their histogenesis and classification, as well as the selection of morphological features determining the malignancy. Despite this, the histopathological examination remains the basic diagnostic procedure in the pathology of mammary gland in animals. Until recently, routine microscopic assessment of surgically removed tumours was based mainly on the morphology of the neoplastic tissue. Over the last few years, progress that has been achieved through implementation of immunohistochemical methods, and molecular biology techniques are beginning to reveal the mechanisms behind carcinogenesis. The indicators of abnormal cell processes discovered during the above studies have been helpful in indentifying and differentiating individual types of neoplastic proliferation. These indicators may be used as prognostic and predictive factors in the treatment of individual carcinoma types, after previously assessing their values.
The aim of this study was to assess the usefulness of expression level of proliferating cell nuclear antigen (PCNA), Ki-67 antigen, the argyrophilic nucleolar organizer regions (AgNORs) count number in the cell nucleus and the intensity of apoptosis when differentiating between non-malignant and malignant tumours of the mammary gland in female dogs. Also considered important was to determine the prognostic value of histologic classification taking into account the assessment of malignancy stage, intensity of cellular proliferation based on expression of PCNA, Ki-67 antigen and AgNORs count in the cell nucleus and the intensity of apoptosis and p53 accumulation in relation to later disease outcome after surgical removal of mammary malignant carcinoma in bitches.
The study materials were 139 mammary tumours collected from selected female dogs either during a mammectomy procedure or a partial or unilateral mastectomy procedure. These tumours were then pathomorphologically assessed based on the clinical-pathological TNM system and histopathological method, including evaluation of the histological malignancy stage. The animals were monitored for 24 months following surgery, and during this period the health of the animals was assessed on the basis of an established schedule of clinical observation, chest radiography and abdomen ultrasonography, recording the time of metastasis diagnosis and/or local recurrence and survival rate. 
During the first stage of the study the diagnostic values of proliferation and apoptosis were assessed, based on the differences in values of the studied parameters between the individual histological types of tumours. The second stage of the study concerned the assessment of prognostic value, for which 103 animals with malignant carcinomas were selected and divided into two groups: group I included 41 animals that had survived the 24-month follow-up period without recurrences and metastases, while group II included 62 animals with an observed progression of neoplastic disease. On the basis of the percentage of animals with disease remission and a disease-free period, the prognostic values of the studied parameters were assessed for both groups. The immunohistochemical method was used to determine the expression of PCNA, Ki-67 antigen and p53 accumulation; the histochemical method was used for AgNORs count; and a modified TUNEL method was used for the evaluation of apoptosis intensity, taking advantage of computer-assisted image analysis for microscopic assessment of the studied parameters.
The results of the studies have shown that, of the studied parameters, the determination of expression of Ki-67 antigen and PCNA was the most useful in differentiating between non-malignant and malignant tumours. The same parameters may be useful to limited degree in differentiating those cases that are problematic in terms of diagnosis during routine histopathological assessment. 
Analysis of the results of the surgical procedure after the 24-month follow-up period has revealed that osteosarcoma and anaplastic carcinoma are the histopathological types with the poorest prognosis. In the case of epithelial carcinomas, the prognosis degrades in the following sequence: non-invasive carcinoma, complex carcinoma, tubular/papillary carcinoma, solid carcinoma and anaplastic carcinoma. Additionally, it was shown that histological malignancy stage significantly influences the risk of disease progression onset and remission period. The results indicate that the assessment of the expression of Ki-67 antigen and PCNA has a high value in predicting the disease-free period. Moreover, the Ki-67 antigen is better than PCNA in assessing the disease progression risk, and its determination is negligibly dependent on the immunohistochemical procedure. This indicates a higher value of determination of Ki-67 antigen expression, in terms of the practical aspect and the possibility of introducing it into routine histopathological diagnostics. Based on these studies, the evaluation of average AgNORs count in the cell nucleus and intensity of apoptosis does not have a prognostic value for mammary tumours in female dogs. The results, although not completely unequivocal, indicate a potential for using the assessment of p53 protein accumulation as a prognostic index. However, because of the risk of false-positive or negative results, this method can not be recommended for routine histopathological diagnostics. The studies have confirmed the high prognostic value of assessment of tumour size according to TNM classification; and although the presence of neoplastic cells in surgically removed lymph nodes was related to a more unfavourable disease course, the observed differences regarding the risk of disease progression and remission period were not statistically significant.
Based on these results, it can be stated that the following parameters are valuable prognostic factors in malignant mammary tumours in bitches: histological type, histological malignancy stage, tumour size according to TNM classification and indices of Ki-67 antigen and PCNA.