Demonstration of silver-stained nucleolar organizer region associated proteins (AgNORs) after wet autoclave pretreatment in breast carcinoma: correlation to tumor stage and long-term survival.

Argyrophilic nucleolar organizer region associated proteins (AgNORs) are known to reflect cellular and nucleolar activity. Due to a novel staining procedure, which substantially improves visualisation of AgNORs on formalin-fixed and paraffin-embedded material, AgNORs can be reliably demonstrated as true substructures of the nucleoli. The aim of the present study was to apply a standardized morphometric AgNOR quantification on a large series of breast carcinomas with regard to its prognostic relevance. AgNOR quantity was evaluated on archival tumor tissues of 115 adenocarcinomas of the breast treated with the wet autoclave method prior to standardized silver-staining and morphometric analysis. AgNOR parameters were correlated to prognostic features (steroid hormonal receptor status, tumor type, tumor size, histological grading, pTNM, and UICC stage) carrying out both univariate and multivariate survival analyses. AgNOR number and area were proven to be statistically significantly related (Pearson correlation coefficient: 0.67, Bonferroni adjusted P = 0.0001). Almost all AgNOR parameters, in particular CV (coefficient of variation) of corrected area (delta-area) and CV of number, were statistically significantly correlated to estrogen and progesterone receptor status as well as histological grading of tumors. Increased AgNOR parameters were statistically significantly associated with early tumor relapse and cancer related death. Univariate and multivariate analysis by means of Cox regression revealed independent prognostic significance for CV of delta-area and number of AgNORs. Various AgNOR parameters (CV of number, CV of delta-area, CV of area, mean delta-area, and mean area of AgNORs per nucleus) determined on wet autoclave pre-treated formalin-fixed and paraffin-embedded breast cancer tissues are statistically highly significantly associated with the prognostic outcome, independently predicting tumor-free and overall survival.