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

Abstract

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.

Standardized AgNOR analysis of the invasive tumour front in oral squamous cell carcinomas

“…Furthermore, the introduction of the coefficient of variation (CV) of simultaneously measured number and area of AgNORs per nucleus provides a highly reproducible parameter of AgNOR analysis. 36 With regard to clinical significance, the outstanding performance of standardized AgNOR analysis in predicting the clinical course of the respective patients independently from established prognostic factors has been proven so far in colorectal, 37 breast, 38 and lung cancer, 39 as well as in OSCCs (if assessed at the invasive tumour front), as demonstrated in the present and previous works. 23,40 Finally, the cost of silver staining is lower than that of most immunohistochemical reactions generally used in tumour diagnostics.…”

Prognostic value of histobiological factors (malignancy grading and AgNOR content) assessed at the invasive tumour front of oral squamous cell carcinomas

“…In the present study, an independent prognostic relevance of the pT stage could be shown exclusively with regard to the prediction of locoregional recurrences ( Table 2). The usefulness of standardized AgNOR parameters as independent predictors of prognosis has been demonstrated recently in different human malignancies (Ofner et al, 1995b;Totsch et al, 1995;Ofner et al, 1996). In a previous study, we could show a significant increase of the AgNOR content at the invasive front of British Journal of Cancer (1997) oral squamous cell carcinomas compared with central parts of the tumours and non-tumorous mucosa (Piffko et al, 1996).…”

“…AgNORs are considered to reflect biosynthetic and nucleolar activity of a cell and thus serve as indicators of the rapidity of the cell cycle (Trere et al, 1989;Derenzini et al, 1994). Using a recently introduced standardized silver staining and morphometric analysis in archival histological material (Ofner et al, 1994(Ofner et al, , 1995a, the independent prognostic value of AgNORs has been established in colonic, lung and breast cancer (Ofner et al, 1995b;Totsch et al, 1995;Ofner et al, 1996).…”

Improvement of breast cancer prognostication using cell kinetic-based silver-stainable nucleolar organizer region quantification of the MIB-1 positive tumor cell compartment

“…As recently reviewed, also the analysis of the silver-stainable nucleolar organizer regions (AgNORs) has been frequently found to be prognostically useful [35]. The standardization of the conventional staining procedure [37] by the introduction of wet autoclaving in 1994 [32,31] has especially improved the meaning of AgNOR analysis, and different studies on the prognostic significance of this new staining protocol have been reported since then on carcinoma of the breast [34], oral cavity [36], lung [48], stomach [17,39], prostate [9], and colorectum [16,33]. However, the analysis of either the MIB-1 immunoreactivity or the amounts of AgNOR describes a static situation only.…”